Anti-leukocyte adhesion for the mitigation of potential adverse events caused by CD3-specific binding domains

ABSTRACT

The present invention relates in essence to use of a compound, which decreases or inhibits the binding of mammalian T-cells to mammalian endothelial cells for use in a method of prophylaxis and/or amelioration and/or treatment of clinical adverse events caused by a therapy which comprises re-directing of T-cells against target cells in a patient. Such a therapy includes, but is not limited to, treatment with an antibody comprising a CD3 binding domain, such as a CD20×CD3 or a CD19×CD3 bispecific single chain antibody, e.g., blinatumomab (MT-103). Methods of treatment of patients having or being at risk of clinical adverse events caused by therapy which comprises re-directing of T-cells against target cells are also contemplated, as are methods of identifying a compound for administration in the methods of prophylaxis, amelioration and/or treatment. Such anti-adhesive type compounds include, but are not limited to, antibodies, like natalizumab, efalizumab, and etrolizumab; minocycline, (acetyl-)salicyclic acid, astilbin, and flavonoids; and thrombin and pentosanpolysulfate (PPS), or a pharmaceutically acceptable salt thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of Provisional U.S. PatentApplication No. 61/762,718, filed Feb. 8, 2013 and Provisional U.S.Patent Application No. 61/811,526, filed Apr. 12, 2013.

The entire contents of the ASCII text entitled “48363_seq_Listing.txt,”created on Feb. 8, 2014, and having a size of 16 kilobytes isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates in essence to a compound which decreasesor inhibits the binding of mammalian T-cells to mammalian endothelialcells for use in a method of prophylaxis and/or amelioration and/ortreatment of clinical adverse events caused by therapy which comprisesre-directing of T-cells against target cells in a patient. Methods oftreatment of patients having or being at risk of clinical adverse eventscaused by therapy which comprises re-directing of T-cells against targetcells are also contemplated. Moreover, this invention relates to kitswhich comprise any of these compounds or a combination thereof, aCD3-specific binding domain or a nucleic acid which encodes a chimericantigen receptor (CAR), and an enclosed label or package insertindicating that the compound or the combination is to be employed forthe prophylaxis or amelioration of clinical adverse events caused by atherapy which comprises re-directing of T cells against target cells.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. To the extent the material incorporated byreference contradicts or is inconsistent with this specification, thespecification will supersede any such material. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

BACKGROUND OF THE INVENTION

In 2012, B cell-malignancies constituted approximately 5% of newlydiagnosed cancers in the US. Age-adjusted incidence rates for acutelymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), andB-cell non-Hodgkin lymphoma (B-NHL) were 1.6, 4.2, and 16.5 per 100,000men and women per year, respectively (Howlader N, Noone A M, Krapcho M,Neyman N, Aminou R, Altekruse S F, Kosary C L, Ruhl J, Tatalovich Z, ChoH, Mariotto A, Eisner M P, Lewis D R, Chen H S, Feuer E J, Cronin K A(eds). SEER Cancer Statistics Review, 1975-2009 (Vintage 2009Populations), National Cancer Institute. Bethesda, Md.,http://seer.cancer.gov/csr/1975_2009_pops09/, based on November 2011SEER data submission, posted to the SEER web site, 2012). Despiterepeated intensive standard treatments B cell-malignancies may becomerefractory to or relapse after therapy and frequently remain incurable.Therefore, a high medical need exists for innovative treatmentmodalities to improve the outcome in these patient populations.

Antibody-based cancer therapies require a target antigen firmly bound tothe surface of cancer cells in order to be active. By binding to thesurface target, the antibody can directly deliver a deadly signal to thecancer cell or indirectly by, for example, recruiting a cytotoxic Tcell, if it is a bispecific antibody. In an ideal treatment scenario, atarget antigen is abundantly present and accessible on every cancer celland is absent, shielded or much less abundant on normal cells. Thissituation provides the basis for a therapeutic window in which a definedamount of the antibody-based therapeutic effectively hits cancer cellsbut spares normal cells.

Monoclonal antibodies were first added to standard chemotherapy about 20years ago, yet this combination has not proved to be completely curativein B cell-malignancies. In recent years, a novel therapeutic approachwith bispecific single-chain antibodies has entered clinical studies andshown promising initial results. Multispecific antibodies, such asbispecific antibodies, which re-direct T-cells, are of special interestfor the treatment of cancer target cells. Re-directing of T cellscomprises that T cells are equipped with an antigen receptor specificitywhich differs from the T cells' clonotypic natural antigen receptorspecificity, i.e. the re-directed T-cells comprise for example a bindingdomain recognizing said cancer target cells. This can e.g. be achievedby T cell engaging bi- or multi-functional antibodies or antibodyderivatives, such as bispecific antibodies comprising inter alia aCD3-specific binding domain, or by transduction of T cells with chimericantigen receptors (CARs) such as CARs recognizing CD19 (seeKnochenderfer et al., Nature Reviews 2013; Clinical Oncology; “TreatingB-cell cancer with T cells expressing anti-CD19 chimeric antigenreceptors”). Bispecific antibodies targeting CD19 or CD20 on B cells andCD3 on T cells are of special interest for the treatment of Bcell-malignancies. Blinatumomab (sometimes also denoted as AMG 103 orMT103) is a recombinant CD19×CD3 bispecific scFv antibody consisting offour immunoglobulin variable domains assembled into a single polypeptidechain. Two of the variable domains form the binding site for CD19 whichis a cell surface antigen expressed on most normal and malignant Bcells. The other two variable domains form the binding site for CD3which is part of the T cell-receptor complex on T cells. By binding toCD19 on a normal or malignant B cell and concomitantly engaging a T cellvia CD3, Blinatumomab induces the formation of a cytolytic synapse(Offner et al. Mol. Immunol. 2006; 43:763-71), thereby leading to theeradication of the bound B cell. Blinatumomab is designed topolyclonally redirect the body's cytotoxic T cells against multiple Btumor cells.

Various clinical studies evaluating the safety and efficacy ofBlinatumomab have been conducted both in B-NHL (Bargou et al. Science.2008; 321:974-7) and B-precursor ALL (Topp et al. J Clin Oncol. 2011;29:2493-8). These studies established clinical proof of concept for thehigh therapeutic potential of the bispecific single-chain antibodyformat in general and of Blinatumomab in special and validated itsfurther development in B-NHL, ALL and CLL.

Though antibodies are an effective means in treating many disorders, inparticular cancer, their administration is not necessarily devoid ofside effects. A “side effect” which is sometimes also denoted as“adverse effect” or more frequently as “adverse event” (sometimes alsodenoted as “AE”) in clinical studies, is a harmful and undesired effectresulting from medication in the treatment of a patient with are-directed T-cell for example by way of a multispecific antibody ormore preferably a bispecific antibody comprising a CD3-specific bindingdomain. Adverse effects may cause a reversible or irreversible change inthe health status of a patient. As adverse effects could be harmful andpotentially even life-threatening it is highly desirable to avoid them.

However, it is difficult to design a therapy which comprisesre-directing of T-cells against target cells in a patient (for example aCD19×CD3 bispecific single-chain antibody-based therapy) which does notcause neurological symptoms, or to put it differently, it is desired toprovide such a therapy, for example a CD19×CD3 bispecific single-chainantibody-based therapy with increased patient tolerability, i.e. reducedor even no harmful side effects such as CNS AEs. It is particularlydesired to mitigate CNS AEs to an extent avoiding discontinuation oftreatment due to CNS AEs thus allowing the patients to fully benefitfrom the treatment.

There is thus a strong need in the art to provide means and methodswhich attenuate or even avoid the above mentioned side effects thattypically accompany a therapy that is based on re-directed T-cells (suchas a therapy that makes use of a CD19×CD3 bispecific single-chainantibody).

The present invention addresses this need and thus provides, as asolution to the technical problem, a compound which decreases orinhibits the binding of mammalian T-cells to mammalian endothelial cellsfor use in a method of prophylaxis and/or amelioration and/or treatmentof clinical adverse events caused by therapy which comprisesre-directing of T-cells against target cells in a patient.

Further embodiments of the present invention are characterized anddescribed herein and also reflected in the claims.

SUMMARY OF THE INVENTION

The present invention provides a compound which decreases or inhibitsthe binding of mammalian T-cells to mammalian endothelial cells for usein a method of prophylaxis and/or amelioration and/or treatment ofclinical adverse events caused by therapy which comprises re-directingof T-cells against target cells in a patient.

In one embodiment, said compound is a compound which

-   -   (a) is capable of binding to a T-cell adhesion molecule,    -   (b) is capable of blocking the binding site of a T-cell adhesion        molecule, and/or    -   (c) inhibits or reduces the expression of a T-cell adhesion        molecule,        and which decreases or inhibits the binding of mammalian T-cells        to mammalian endothelial cells, for use in a method of        prophylaxis and/or amelioration and/or treatment of clinical        adverse events caused by therapy which comprises re-directing of        T-cells against target cells in a patient.

In a further embodiment, said compound is a compound which

-   -   (a) is capable of binding to an endothelial adhesion molecule,    -   (b) is capable of blocking the binding site of an endothelial        adhesion molecule, and/or    -   (c) inhibits or reduces the expression of an endothelial        adhesion molecule,        and which decreases or inhibits the binding of mammalian T-cells        to mammalian endothelial cells, for use in a method of        prophylaxis and/or amelioration and/or treatment of clinical        adverse events caused by therapy which comprises re-directing of        T-cells against target cells in a patient.

The present invention also provides a method of identifying a compoundfor use in a method of prophylaxis and/or amelioration and/or treatmentof clinical adverse events caused by therapy which comprisesre-directing of T-cells against target cells in a patient, comprising:

-   -   (a) contacting said compound with a mammalian T-cell, a        mammalian endothelial cell, a T-cell adhesion molecule and/or an        endothelial adhesion molecule; and    -   (b) evaluating whether said compound:        -   (i) decreases or inhibits the binding of mammalian T-cells            to mammalian endothelial cells;        -   (ii) is capable of binding to a T-cell adhesion molecule,        -   (iii) is capable of blocking the binding site of a T-cell            adhesion molecule,        -   (iv) inhibits or reduces the expression of a T-cell adhesion            molecule,        -   (v) is capable of binding to an endothelial adhesion            molecule,        -   (vi) is capable of blocking the binding site of an            endothelial adhesion molecule, and/or        -   (vii) inhibits or reduces the expression of an endothelial            adhesion molecule.

It is envisaged in one embodiment that said therapy which comprisesre-directing of T-cells against target cells in a patient, includes aCD3 binding domain.

In another embodiment it is envisaged that said therapy which comprisesre-directing of T-cells against target cells in a patient, includes agenetically engineered T-cell having a chimeric antigen receptor (CAR).

It is also envisaged that the compound which is to be used in accordancewith the present invention, is to be administered prior to and/orconcomitantly with an initial dosing, a re-exposure (i.e. a re-dosing,such as for example a subsequent dosage step) and/or an increase of thedosage of said therapy (which is common practice when administering forexample an antibody) which comprises re-directing of T-cells againsttarget cells in a patient.

The present invention further provides a kit comprising a compound asdefined or identified in accordance with the present invention, togetherwith a CD3 binding domain.

The present invention also provides a kit comprising a compound asdefined or identified in accordance with the present invention, togetherwith a nucleic acid which encodes a chimeric antigen receptor (CAR).

A pharmaceutical composition comprising a compound as defined oridentified in accordance with the present invention together with a CD3binding domain.

The present invention further relates to a CD3 binding domain for use ina method of re-directing of T-cells against target cells in a patient,wherein said patient is subject to therapy comprising the compound asdefined herein.

In one embodiment, the present invention relates to a nucleic acid whichencodes a chimeric antigen receptor (CAR) for use in a method ofre-directing of T-cells against target cells in a patient, wherein saidpatient is subject to therapy comprising the compound as defined herein.

In one embodiment said clinical adverse events comprise neurologicaladverse events.

In a further embodiment, said neurological adverse event is/are one ormore of (i) cognitive disorder comprising disorientation/confusionand/or word finding problems/aphasia, (ii) seizure, (iii) cerebellarsymptoms partly observed as an optional prodromal phase of (i) or (ii)comprising kinetic tremor, ataxia, dysarthria and handwriting problems.

In the context of the present invention, it is particularly envisagedthat said CD3 binding domain is a bispecific single chain antibody.

Said bispecific single chain antibody comprises in a further embodimentof the present invention a binding domain which is specific for B-cells,preferably specific for a CD-marker that can be found on B-cell lymphomasuch as CD19, CD22, CD20 or CD79a, CD19 being preferred.

In one embodiment, said bispecific single chain antibody is a CD19×CD3or CD20×CD3 bispecific single chain antibody.

In further embodiments of the present invention, said chimeric antigenreceptor (CAR) comprises a binding domain which is specific for B-cells,preferably specific for a CD-marker that can be found on B-cell lymphomasuch as CD19, CD22, CD20 or CD79a, CD19 being preferred.

In a particularly preferred embodiment of the present invention, saidCD19×CD3 bispecific single chain antibody is Blinatumomab (sometimesalso denoted as MT103 or AMG103).

In a further preferred embodiment of the present invention, said patientis characterized by a B/T-cell ratio of less than 1:5 or a B-cell numberof less than about 50 B-cells per μl peripheral blood.

It is envisaged that the compound that is to be used in the context ofthe present invention is selected from the compounds disclosed herein.

The present invention also relates to a method for prophylaxis,amelioration and/or treatment of clinical adverse events caused bytherapy which comprises re-directing of T-cells against target cells ina patient, said method comprising administering a therapeuticallyeffective amount of a compound defined herein.

In one embodiment of the present invention, said mammalian T-cells,whose binding to mammalian endothelial cells is decreased or inhibitedby the compound defined herein, are re-directed mammalian T-cells.

It is also contemplated that the mammalian endothelial cells mentionedin the context of the present invention as described herein, areisolated from large blood vessels or capillaries.

In one embodiment, said mammalian endothelial cells are selected fromHuman Umbilical Vein Endothelial Cells (HUVEC) or Human BrainMicrovascular Endothelial Cells (HBMEC), HBMECs being preferred.

The T-cell adhesion molecule is, in the context of the presentinvention, selected from integrins (such as alpha4-integrins;alphaL-beta2-integrins, alphaL-integrins, beta7-integrins), selectins(such as L-selectin), and/or CD44.

The endothelial adhesion molecule is, in the context of the presentinvention, selected from selectins (such as E- or P-selectin); celladhesion molecules CAMs (such as ICAM-1, MAdCAM, VCAM-1) and/or PAR-1.

It is also envisaged that in the context of the present invention, saidpatient is a mammal, preferably a primate, and most preferably a humanbeing.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Exemplary time courses of selected pharmacodynamic markersduring treatment week 1 of a B-NHL patient treated at a Blinatumomabdose level of 60 μg/m²/day in a phase 1 clinical study.

Cell counts/percentage and protein concentration in peripheral blood andserum are shown, respectively. A. T cell-redistribution characterized bya rapid T cell-disappearance from peripheral blood immediately (i.e.already at 45 minutes) after start of infusion. B. Increased Tcell-numbers with intermediate-affinity (i.e. activated) LFA-1 on itscell surface peaking concurrently with T cell-redistribution. C.Increased serum concentrations of Ang-2 released by activatedendothelial cells during T cell-redistribution. D. T cell-redistributionis accompanied by disappearance of other mononuclear cells (as shown fore.g. monocytes) from peripheral blood with prolonged recovery tobaseline counts.

FIG. 2. Current hypothesis of the multi-step pathomechanism leading tocentral nervous system adverse events caused by Blinatumomab.

A. Start of infusion or stepwise dose increase of Blinatumomab increaseT cell-adhesion to blood vessel endothelium. B. Adherent T cellsactivate the endothelium and start to extravasate. Activated endothelialcells attract other peripheral blood leukocytes, e.g. monocytes.Extravasated T cells secrete cytokines and chemokines which in turncause transient neuroinflammation and pertubation of theblood-CSF-barrier.

FIG. 3. Delayed T cell-redistribution kinetics in three patients whoreceived PPS co-medication for the prophylaxis or amelioration of CNSAEs and the prevention of treatment discontinuations due to said CNS AEs(D, E, and F) compared to patients who did not receive PPS co-medication(A, B, and C).

T and B cell-counts are shown as absolute numbers per μl of peripheralblood. The time axis gives the period within the respective treatmentcycle. Blinatumomab dose levels, e.g. 5, 15, or 60 μg/m²/day areindicated. The time points 0 h, 45 min, and 2 h after start of infusionor any dose step revealing differences in T cell-redistribution kineticsare indicated.

FIG. 4. Comparison of flow-cultivated versus statically cultivatedendothelial cells.

A. HBMEC were cultivated in μ-slide Luer I^(0.4) Collagen IV for 48 hunder wall shear stress of 5 dyn/cm² (left) or under static conditions(right). B. HUVEC were cultivated in μ-slide Luer I^(0.4) ibiTreat for48 h under wall shear stress of 10 dyn/cm² (left) or under staticconditions (right). Microscopic analysis was done with a 10× objective.

FIG. 5. Influence of AMG 103 on T cell-rolling and adhesion on HUVECunder short-term conditions.

Freshly isolated T cells were pre-incubated with or without AMG 103 (10ng/ml) for 40 min at 37° C. prior to rolling of T cells onflow-cultivated HUVEC for 45 sec at shear stress of 1 dyn/cm². Rollingvelocities and absolute numbers of adherent cells were determinedmicroscopically by using the automated tracking module. A. Datarepresent the mean±SD T cell-rolling velocity. ***: P<0.001. B. Datarepresent absolute numbers of adherent T cells. C. Immunofluorescencestaining of PFA-fixed HUVEC after rolling of T cells pre-incubated with(+AMG 103) or without (−AMG 103) AMG 103 at shear stress of 1 dyn/cm²for 45 sec. Microscopic analysis was done with a 20× objective andUV-light. ICAM-1 and P-selectin staining is shown in red and green,respectively.

FIG. 6. Influence of AMG 103 on T cell-rolling on HBMEC under long-termconditions.

A. Freshly isolated T cells were used with or without addition of AMG103 (10 ng/ml) for rolling on flow-cultivated HBMEC for 120 min at shearstress of 1 dyn/cm². Image acquisitions were performed immediately afterstart of rolling (0 min) and after 45 min of continuous rolling onHBMEC. Rolling velocities were determined microscopically by using theautomated tracking module. Data represent the mean±SD T cell-rollingvelocity. ***: P<0.001; ns: P>0.05 (not significant). B.Immunofluorescence staining of PFA-fixed HBMEC after T cell-rolling inthe presence (+AMG 103) or absence (−AMG 103) of AMG 103 for 120 min atshear stress of 1 dyn/cm². Microscopic analysis was done with a 20×objective and UV-light. ICAM-1, P-selectin, and VCAM-1 staining is shownin red, green, and blue, respectively.

FIG. 7. Influence of PPS on T cell-rolling on histamine-stimulated HBMECunder long-term conditions.

A. Rolling of freshly isolated T cells on histamine-stimulated,untreated or PPS pre-incubated (200 μg/ml), flow-cultivated HBMEC wasperformed with or without AMG 103 (10 ng/ml) for 60 min of continuousrolling at shear stress of 1 dyn/cm². Rolling velocities were determinedmicroscopically by manually tracking 30 T cells for each condition. Datarepresent the mean±SD T cell-rolling velocity at time point 45 min. ***:P<0.001; ns: P>0.05 (not significant). B. Immunofluorescence staining ofPFA-fixed histamine-stimulated, untreated (—PPS) or PPS pre-incubated(+PPS) HBMEC after T cell-rolling in the presence (+AMG 103) or absence(−AMG 103) of AMG 103 for 60 min at shear stress of 1 dyn/cm².Microscopic analysis was done with a 20× objective and UV-light.P-selectin staining is shown in green.

FIG. 8. Influence of PPS on T cell-rolling on non-stimulated HBMEC underlong-term conditions.

Continuous rolling of freshly isolated T cells on non-stimulated,untreated or PPS pre-incubated (200 μg/ml), flow-cultivated HBMEC wasperformed with or without AMG 103 (10 ng/ml) for 60 min at shear stressof 1 dyn/cm². Rolling velocities were determined microscopically bymanual tracking of 30 T cells for each condition. Data represent themean±SD T cell-rolling velocity at time point 40 min. ****: P<0.0001;***: P<0.001; **: P<0.01; ns: P≧0.05 (not significant).

FIG. 9. Influence of natalizumab on T cell-rolling on non-stimulatedHBMEC under long-term conditions.

Continuous rolling of freshly isolated T cells pre-incubated with orwithout natalizumab (1 μg/ml) for 10 min at 37° C. on non-stimulated,flow-cultivated HBMEC was performed with or without AMG 103 (10 ng/ml)for 60 min at shear stress of 1 dyn/cm². Rolling velocities weredetermined microscopically by manual tracking of 30 T cells for eachcondition. Data represent the mean±SD T cell-rolling velocity at timepoint 40 min. ***: P<0.001; *: P<0.05; ns: P≧0.05 (not significant).

FIG. 10. Influence of minocycline on T cell-rolling on non-stimulatedHUVEC under long-term conditions.

Continuous rolling of freshly isolated T cells pre-incubated with orwithout minocycline (50 μg/ml) for 2 h at 37° C. on non-stimulated,flow-cultivated HUVEC was performed with or without AMG 103 (10 ng/ml)for 45 min at shear stress of 1 dyn/cm². Rolling velocities weredetermined microscopically by using the automated tracking module. Datarepresent the mean±SD T cell-rolling velocity at time point 40 min.****: P<0.0001; ns: P≧0.05 (not significant).

FIG. 11. Influence of minocycline on T cell-adhesion to non-stimulatedHUVEC under long-term conditions.

Continuous rolling of freshly isolated T cells pre-incubated with orwithout minocycline (50 μg/ml) for 2 h at 37° C. on non-stimulated,flow-cultivated HUVEC was performed with or without AMG 103 (10 ng/ml)for 45 min at shear stress of 1 dyn/cm². Absolute numbers of adhering Tcells were determined microscopically by manual cell counting. Datarepresent the absolute number of adhering T cells per image section attime point 40 min.

FIG. 12. Influence of an anti-ICAM-1 antibody on T cell-rolling onnon-stimulated HBMEC under long-term conditions.

Continuous rolling of freshly isolated T cells on non-stimulated,untreated or with mouse anti-human ICAM-1 antibody pre-incubated (10μg/ml), flow-cultivated HBMEC was performed with or without AMG 103 (10ng/ml) for 45 min at shear stress of 1 dyn/cm². Rolling velocities weredetermined microscopically by manual tracking of 30 T cells for eachcondition. Data represent the mean±SD T cell-rolling velocity at timepoint 30 min. ***: P<0.001; **: P<0.01; ns: P≧0.05 (not significant).Ab: antibody.

FIG. 13. Influence of an anti-P-selectin antibody on T cell-rolling onnon-stimulated HBMEC under long-term conditions.

Continuous rolling of freshly isolated T cells on non-stimulated,untreated or with mouse anti-human P-selectin antibody pre-incubated (10μg/ml), flow-cultivated HBMEC was performed with or without AMG 103 (10ng/ml) for 45 min at shear stress of 1 dyn/cm². Rolling velocities weredetermined microscopically by manual tracking of 30 T cells for eachcondition. Data represent the mean±SD T cell-rolling velocity at timepoint 40 min. **: P<0.01; *: P<0.05; ns: P≧0.05 (not significant). Ab:antibody.

FIG. 14. Influence of an anti-CD11a antibody on T cell-rolling onnon-stimulated HBMEC under long-term conditions.

Continuous rolling of freshly isolated T cells pre-incubated with orwithout anti-CD11a antibody (5 μg/ml) for 10 min at 37° C. onnon-stimulated, flow-cultivated HBMEC was performed with or without AMG103 (10 ng/ml) for 45 min at shear stress of 1 dyn/cm². Rollingvelocities were determined microscopically by using the automatedtracking module. Data represent the mean±SD T cell-rolling velocity attime point 10 min. ****: P<0.0001; ns: P≧0.05 (not significant). Ab:antibody.

FIG. 15. Influence of an anti-CD 162 antibody on T cell-rolling onnon-stimulated HBMEC under long-term conditions.

Continuous rolling of freshly isolated T cells pre-incubated either withor without anti-CD162 antibody (10 μg/ml) or with mouse isotype controlantibody (10 μg/ml) for 10 min at 37° C. on non-stimulated,flow-cultivated HBMEC was performed with or without AMG 103 (10 ng/ml)for 45 min at shear stress of 1 dyn/cm². Rolling velocities weredetermined microscopically by manual tracking of 30 T cells for eachcondition. Data represent the mean±SD T cell-rolling velocity at timepoint 45 min. ****: P<0.0001; *: P<0.05; ns: P≧0.05 (not significant).Ab: antibody.

FIG. 16. Influence of recombinant P-selectin on T cell-rolling onnon-stimulated HBMEC under long-term conditions.

Continuous rolling of freshly isolated T cells pre-incubated with orwithout recombinant P-selectin (5 μg/ml) for 15 min at 37° C. onnon-stimulated, flow-cultivated HBMEC was performed with or without AMG103 (10 ng/ml) for 45 min at shear stress of 1 dyn/cm². Rollingvelocities were determined microscopically by manual tracking of 40 Tcells for each condition. Data represent the mean±SD T cell-rollingvelocity at time point 45 min. **: P<0.01; *: P<0.05; ns: P≧0.05 (notsignificant). rec.: recombinant.

FIG. 17. Influence of Blinatumomab (AMG 103) on T cell-rolling onrecombinant human adhesion molecules under semi short-term conditions.

Freshly isolated T cells were incubated with or without AMG 103 (10ng/ml) for 35 min at 37° C. prior to continuous rolling with or withoutAMG 103 on recombinant proteins for 15 min at shear stress of 1.1dyn/cm². Rolling velocities were determined microscopically by using theautomated tracking module. Data represent the mean±SD T cell-rollingvelocity at time point t=45 min on A. ICAM-1 (***: P<0.001), B. VCAM-1(***: P<0.001), and at time point t=50 min on C. P-selectin (****:P<0.0001).

Furthermore, Jurkat E6.1 T cells were used as described above instead offreshly isolated T cells. Rolling velocities were determinedmicroscopically by using the automated tracking module. Data representthe mean±SD T cell-rolling velocity at time point t=45 min on D.ICAM-1/VCAM-1 (***: P<0.001), and E. P-selectin (**: P<0.01).

FIG. 18. Influence of Src kinase inhibitor PP2 on T cell-rolling onrecombinant human adhesion molecules under semi short-term conditions.

Freshly isolated T cells pre-incubated with Src kinase inhibitor PP2 (15μM) or vehicle control DMSO for 40 min at 37° C. were further incubatedwith or without AMG 103 (10 ng/ml) in the presence of PP2 or DMSO for 35min at 37° C. prior to continuous rolling on recombinant human VCAM-1for 15 min at shear stress of 1.1 dyn/cm². Rolling velocities weredetermined microscopically by manual tracking of 30 T cells for eachcondition. Data represent the mean±SD T cell-rolling velocity at timepoint t=40 min. ***: P<0.001; ns: P ≧0.05 (not significant).

DETAILED DESCRIPTION OF THE INVENTION

Various clinical studies evaluating the safety and efficacy ofBlinatumomab have been conducted both in B-NHL (Bargou et al. Science.2008; 321:974-7) and B-precursor ALL (Topp et al. J Clin Oncol. 2011;29:2493-8). In B-NHL, doses as low as 0.005 mg/m²/day administered bycontinuous intravenous (civ) infusion over four weeks led to a completeand sustained elimination of B lymphoma cells in peripheral blood.Partial and complete responses were first observed at a dose level of0.015 mg/m²/day and the majority of B-NHL patients treated at a doselevel of 0.06 mg/m²/day experienced a substantial tumor regression.Blinatumomab also led to clearance of malignant B cells from bone marrowand liver in this indication. In B-precursor ALL, patients with bothminimal residual disease and relapsed or refractory disease achieved ahematological complete response when treated with four-week civinfusions at a dose level of 0.015 mg/m²/day. These studies establishedclinical proof of concept for the high therapeutic potential of thebispecific single-chain antibody format in general and of Blinatumomabin special and validated its further development in B-NHL, ALL and CLL.Throughout these studies in B-NHL and B-precursor ALL severalpharmacodynamic markers were assessed. Selected general characteristicsshall be described hereafter: T cell-kinetics displayed a verydistinguished profile irrespective of the dose level or the presence ofcirculating B cells. It was characterized by a swift redistributionafter start of infusion and any dose step, i.e. a fast disappearance ofcirculating T cells within the first 6-12 hours and a subsequentreappearance during the following 2-7 days where high initial Bcell-counts correlated with decelerated kinetics of T cell-reappearance(FIG. 1A). This course seemed to be triggered by any significant dosechanges of Blinatumomab rather than absolute serum concentrations. Inaddition, T cell-adhesiveness was measured throughout treatment week 1by analyzing binding of soluble ICAM-1-F_(c) fusion proteins to LFA-1 onT cells. LFA-1 conformation shifted from a low affinity state beforestart of infusion to an intermediate affinity state after start ofinfusion and any dose step; the increased binding affinity to ICAM-1peaked within 48 hours and returned to baseline within 5 days thereafter(FIG. 1B). This finding coincided with T cell-redistribution andsupported the concept of Blinatumomab-induced T cell-adhesion toendothelium during redistribution.

Angiopoietin-2 (Ang-2) is a serum marker for endothelial activation (oreven endothelial stress) that is stored in so-called Weibel-Paladebodies inside the cytoplasm of blood vessel-lining endothelial cells.Once endothelial cells get activated the vesicles fuse with the cellmembrane and release the preformed Ang-2 into serum. Additionally,vesicle-bound adhesion molecules, e.g. P-selectin thereby appear on thecell surface thus further increasing adhesiveness of blood vessel-liningendothelial cells. Kinetics of Ang-2 serum concentrations resembledLFA-1-mediated increased T cell-adhesiveness by peaking during Tcell-redistribution and declining back to baseline within treatmentweek 1. With maximal Ang-2 serum concentrations coinciding with sound Tcell-disappearance this observation further indicated pronounced Tcell-adhesion to blood vessel-lining endothelial cells (which thus gotactivated) as underlying mechanism of T cell-redistribution (FIG. 1C).

Although not directly engaged by Blinatumomab, monocytes displayedsimilar redistribution kinetics as T cells did. This finding furthersupported the concept of Blinatumomab-induced T cell-binding toendothelium leading to activation of endothelial cells which in turnup-regulated additional adhesion molecules and thereby allowed for broadbinding of other mononuclear cells and thrombocytes (i.e. counts ofcirculating thrombocytes also decreased after start of infusion and anydose step). However, recovery of monocyte-counts in peripheral bloodmight be prolonged as activated (e.g. by adhering to activatedendothelial cells) monocytes tended toward transmigration intounderlying tissue before returning into circulation (FIG. 1D).

Based on the above, the present inventors conceived a hypothesis of apossible multi-step pathomechanism which might lead inter alia tocentral nervous system adverse events (CNS AE) caused by re-directedmammalian T-cells, (e.g. T-cells re-directed by Blinatumomab). Thishypothesis is illustrated in FIG. 2.

FIG. 2A: Start of infusion or stepwise dose increase of Blinatumomabincrease T cell-adhesion to blood vessel endothelium. FIG. 2B: AdherentT cells activate the endothelium and start to extravasate. Activatedendothelial cells attract other peripheral blood leukocytes, e.g.monocytes which in turn cause transient neuroinflammation andperturbation of the blood CSF-barrier.

In order to prove the above hypothesis, the present inventors devised aco-medication scheme wherein Blinatumomab was administered concomitantlywith the heparinoid pentosanpolysulfate (PPS), a small-moleculeinhibitor of P-selectin (Höpfner et al. J Pharm Pharmacol. 2003;55:697-706). Anti-leukocyte adhesion was successfully tested in a phase1 clinical study by transiently infusing patients with PPS, prior to andafter start of infusion of Blinatumomab as well as prior to and afterany stepwise dose increase of Blinatumomab. P-selectin is known tomediate the first step of leukocyte adhesion to endothelial cells andseems to play a particular important role for the extravasation ofcirculating leukocytes via meningeal microvessels to the leptomeningealspace and the meninges (Kivisäkk et al. Proc Natl Acad Sci USA. 2003;100:8389-94).

Three patients received in particular an intravenous PPS infusion duringboth the start-of-infusion phase at 5 μg/m²/day of Blinatumomab and thedose-step phase to 60 μg/m²/day after treatment week 1 (see also theExample section for further details). Despite having a high risk fordeveloping CNS AEs due to their low B:T cell-ratios in the peripheralblood (see the underlying rationale as disclosed in PCT/EP2010/066207),none of these three patients had to discontinue treatment withBlinatumomab due to neurological adverse effects. Thus, much to thesurprise of the present inventors it was indeed possible to mitigate theexpected CNS AE in these patients by way of decreasing the adhesion ofre-directed T-cells to the respective endothelial cells. Two out of thethree patients achieved a complete response after 8 weeks of treatmentwith Blinatumomab; one patient had a stable disease after 4 weeks oftreatment. Moreover, all three patients who had received intravenous PPSfor the mitigation of potential CNS AEs showed delayed Tcell-redistribution kinetics (in the absence of CD19-positive targetcells) upon start of infusion or stepwise dose increase of Blinatumomab(FIG. 3). As described earlier, patients receiving Blinatumomab withoutco-medication with PPS consistently showed a rapid decline of Tcell-counts in the peripheral blood already 45 minutes after start ofinfusion or stepwise dose increase (such as shown in FIGS. 3A, B, andC). By contrast, no decline of T cell-counts compared to the respectivebaseline values was observed 45 minutes after start of infusion orstepwise dose increase in all three patients who had receivedco-medication with PPS during both the start-of-infusion and thedose-step phase (FIGS. 3D, E and F). In two cases (D and E), there waseven an increased T cell-count at 45 minutes. In one case (F), anincreased T cell-count was still observed at 2 hours. The redistributionprocess and thus the underlying leukocyte adhesion to blood vesselendothelium was obviously slowed down by intervention with intravenousPPS as decreased T cell-counts in the peripheral blood were not detectedearlier than 2 hours after start of infusion or stepwise dose increaseof Blinatumomab.

Thus, a pharmacodynamic marker (i.e. delayed T cell-redistributionkinetics) for the prognosticated and intended mode of action of PPS wasidentified in patients. The clinical courses of the patients whoreceived co-medication with PPS are consistent with these biomarkerobservations and also with the predictions of the current hypothesis onthe pathomechanism of central nervous system adverse events.Accordingly, interfering with leukocyte adhesion to blood vesselendothelium (i.e. anti-leukocyte adhesion) is a mechanism-basedinterventional approach to prevent or ameliorate side effects such asCNS AEs caused by therapy which comprises re-directing of T-cellsagainst target cells in a patient, such as Blinatumomab (AMG 103).

To further investigate and confirm that the initial binding ofre-directed (human) T-cells to the respective (human) endothelial cellsis indeed causative for the above mentioned CNS AE, the presentinventors additionally established a test system which simulatesrolling, tethering and adhesion of re-directed (human) T cells and otherleukocytes on/to (human brain) microvascular endothelial cells underhydrodynamic flow conditions in an in vitro system (“flow system”).Further details of this test system can be derived from the appendedexamples which explain the experimental setup in great and sufficientdetail (see the Example section). In this flow system the cellularrolling velocity as well as the number of adherent cells can be easilymeasured by those skilled in the art. Interference with any (molecular)step (i.e. requirement) of the multi-step rolling and adhesion processof peripheral blood cells on/to endothelial cells is expected toinfluence both the cellular rolling velocity as well as the number ofadherent cells.

Addition of a CD3-specific binding domain, in particular Blinatumomab,to the flow system (thereby creating a re-directed T-cell), rapidly andsignificantly decreased T cell-rolling velocity on human brainmicrovascular endothelial cells (HBMEC). Simultaneously, HBMEC gotactivated by these re-directed T-cells as shown by up-regulation ofadhesion molecules P-selectin, ICAM-1 and VCAM-1 on their cell surface(see FIG. 6B). However, the addition of T-cells without Blinatumomab(resulting in non-re-directed T-cells) did not influence the expressionof the aforementioned endothelial adhesion molecules (see FIG. 6B).These observations resemble and correspond unequivocally to the Tcell-redistribution as seen in patients and described in detail herein.Moreover, addition of the P-selectin blocking agent PPS to the flowsystem (i.e. pre-incubation of HBMEC with PPS) could efficiently blockBlinatumomab-induced reduction of T cell-rolling velocity on HBMEC. Asdemonstrated after addition of Blinatumomab to the flow system,pre-incubation with PPS could neutralize any Blinatumomab-inducedeffects and revert T cell-rolling velocity back to levels comparable tothose observed for T cell-rolling velocity without the addition ofBlinatumomab to the flow system. Additionally, HBMEC got less activatedwhen PPS was present in the flow system as shown by reduced cell surfaceexpression of P-Selectin. These observations support the clinicalfinding that the addition of a compound which decreases or inhibits thebinding of mammalian T-cells to mammalian endothelial cells (such as theP-selectin blocking agent PPS) is indeed able to decrease/delay Tcell-adhesion to endothelial cells and thereby to prevent, ameliorationand/or treatment clinical adverse events caused by a therapy whichtherapy comprises re-directing of T-cells against target cells in apatient.

Thus, the established flow system truly mimics T cell-rolling andadhesion inter alia of re-directed T-cells on/to blood vessel-liningendothelial cells under hydrodynamic flow conditions as observed duringT cell-redistribution in patients treated with a medicament whichcomprises re-directing of T-cells against target cells, which can beachieved for example with a CD3-specific binding domain such asBlinatumomab. Especially, effects of infusing PPS to patients and addingPPS to the flow system on T cell-redistribution and T cell-rollingvelocity, respectively, are very comparable in that PPS clearlyinterferes with T cell-adhesion to endothelial cells. According to thecurrent hypothesis on the pathomechanism of CNS AEs, this interference(i.e. anti-leukocyte adhesion the decrease or inhibition of the bindingof leucocytes, such as mammalian T-cells, and in particular there-directed T-cells, to mammalian endothelial cells) may treat, preventor ameliorate any adverse side effect, such as CNS AEs, caused by theadministration of a medicament which comprises re-directing of T-cellsagainst target cells to a patient (which can be achieved for example byCD3-specific binding domain or the CARs which are explained hereinelsewhere). Moreover, the established flow system exemplified herein iscapable of and can be used by those skilled in the art in a method ofidentifying a compound for use in a method of prophylaxis and/oramelioration and/or treatment of clinical adverse events caused bytherapy which comprises re-directing of T-cells against target cells ina patient. The exemplified flow system is thus suitable foridentifying/defining compounds with anti-adhesive properties (inparticular compounds that decreases or inhibits the binding of mammalianT-cells to mammalian endothelial cells) that may be administered priorto, concurrently with and/or subsequently to the treatment of a patientwith for example a CD3-specific binding domain for the prophylaxis oramelioration of CNS AEs caused by said CD3-specific binding domain. Inother words, compounds demonstrating any anti-adhesive effects onleukocytes, in particular T cells, and most preferably re-directedT-cells in the flow system (i.e. for example reversion of CD3-specificbinding domain-induced reduction of T cell-rolling velocity on bloodvessel-lining endothelial cells comparable to the observed effects forthe addition of PPS to the flow system) are expected to prevent orameliorate any CNS AEs caused by treatment of a patient with aCD3-specific binding domain when administered prior to, concurrentlywith and/or subsequently to the treatment with said CD3-specific bindingdomain. The identification modification and/or confirmation of suchcompounds, is a straightforward task for the skilled person as it makesuse of a well-established methodology.

The present inventors have thus paved the way to solve the technicalproblems identified herein.

DEFINITIONS

It must be noted that as used herein, the singular forms “a”, “an”, and“the”, include plural references unless the context clearly indicatesotherwise. Thus, for example, reference to “a reagent” includes one ormore of such different reagents and reference to “the method” includesreference to equivalent steps and methods known to those of ordinaryskill in the art that could be modified or substituted for the methodsdescribed herein.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the present invention.

The term “and/or” wherever used herein includes the meaning of “and”,“or” and “all or any other combination of the elements connected by saidterm”.

The term “about” or “approximately” as used herein means within ±20%,preferably within ±15%, more preferably within ±10%, and most preferablywithin ±5% of a given value or range.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step. Whenused herein the term “comprising” can be substituted with the term“containing” or “including” or sometimes when used herein with the term“having”.

When used herein “consisting of” excludes any element, step, oringredient not specified in the claim element. When used herein,“consisting essentially of” does not exclude materials or steps that donot materially affect the basic and novel characteristics of the claim.

In each instance herein any of the terms “comprising”, “consistingessentially of” and “consisting of” may be replaced with either of theother two terms.

The present invention relates in one embodiment to a compound whichdecreases or inhibits the binding of mammalian T-cells to mammalianendothelial cells for use in a method of prophylaxis and/or ameliorationand/or treatment of clinical adverse events caused by therapy whichcomprises re-directing of T-cells against target cells in a patient.

The term “binding” of mammalian T-cells is to be understood to includeany of the well-known sequential steps that typically characterize themovement of leucocytes out of the blood system, a process which isusually denoted as leucocyte extravasation. The aforementioned stepsinclude the “rolling of the leucocytes” (the leucocytes slackly attachto the endothelial cells but are still pulled along with the bloodstream, which results in a rolling motion of the leucocytes on thesurface of the endothelial cells); the “tethering of the leucocytes”(sometimes also denoted as tight attachment—the leucocytes tightlyattach to the endothelial cells, whereas the receptors for thisinteraction are different from those involved in the rolling process)and the “diapedesis step” (sometimes also denoted as transmigration—thepreviously mostly spherical leucocytes spread on the endothelium andactively transmigrate through the endothelial barrier). The compounds ofthe present invention either influence all of these steps, or just someof these steps, or just one of these steps.

The “compound” applied in the present invention (i.e. the compound whichdecreases or inhibits the binding of mammalian T-cells to mammalianendothelial cells) is one which, so to say, acts for example on the Tcell side, i.e., on a T cell adhesion molecule, or on the endothelialcell side, i.e., on an endothelial cell adhesion molecule. However, thecompound may also be one which acts on both the T cell side and theendothelial cell side. Said compound, when applied in the presentinvention, decreases or inhibits the binding of mammalian T cells tomammalian endothelial cells. As such a compound of the present inventionis a compound that effects anti-adhesive effects, either on T cells orendothelial cells or both. For example, a compound may act on both Tcell adhesion molecules and endothelial cell adhesion molecules or amixture of two or more compounds is applied, one acts on T cell adhesionmolecules and the further one(s) on endothelial cell adhesion molecules.An anti-adhesive effect preferably includes that the compound decreasesor inhibits the binding of mammalian T-cells to mammalian endothelialcells. Preferred compounds of the present invention are depicted inTable 1.

It is also envisaged that the compound of the invention, i.e. a compoundwhich decreases or inhibits the binding of mammalian T-cells tomammalian endothelial cells have, is able to increase the mean Tcell-rolling velocity±SD (μm/sec) on HBMEC in the presence of +10 ng/mlAMG 103 by about 30, 40, 50, 60, 70, 80, 90, 100% or even more (seeExample 2—the influence of PPS on Blinatumomab-induced reduction of meanT cell-rolling velocity as observed 45 min after addition of 10 ng/ml ofBlinatumomab to the flow system was evaluated in long-term conditions.While AMG 103 significantly reduced mean T cell-rolling velocity±SD onHBMEC from 430±92 μm/sec (−AMG 103) to 281±96 μm/sec (+AMG 103), furtheraddition of PPS to the flow system reverted this reduction to a mean Tcell-rolling velocity±SD of 483±157 μm/sec as also observed in theabsence of AMG 103 (i.e. −AMG 103, +PPS; 442±156 μm/sec).

It is known that T cells interact with endothelial cells via adhesionmolecules, i.e. T cell adhesion molecules and endothelial cell adhesionmolecules. Both T cell and endothelial cell adhesion molecules belong tothe family of integrins, selectins and immunoglobulin G (IgG)superfamily. The latter are characterized by having an immunglobulindomain. CD44, as receptor of hyaluronic acid being present on thesurface of endothelial cells is also regarded as T cell adhesionmolecule.

Accordingly, preferred compounds that decrease or inhibit the binding ofT cells to endothelial cells are integrins antagonists, selectinantagonists, Ig superfamily cell adhesion molecule antagonists or CD44antagonists, respectively. Thus, a compound applied in the context ofthe present invention is preferably an integrin antagonist, a selectinantagonist, an Ig superfamily cell adhesion molecule antagonist, or aCD44 antagonist, respectively. Any of these antagonists is, in thecontext of the present invention, able to decrease or inhibit binding ofmammalian T cells to mammalian endothelial cells.

Integrin antagonists are commonly known in the art; see e.g. Curley etal. (1999), Cellular and Molecular Life Science 56, 427-441. Selectinantagonists are known in the art; see e.g. Lefer (2010), Ann. Rev.Pharmacol Toxicol 40 283-294. CD44 antagonists are known in the art; seee.g. Hirota-Takahata (2007), J. Antiobiotics 60, 633-639. The same istrue for IgG superfamily cell adhesion molecule antagonists.

A compound that decrease or inhibit the binding of T cells toendothelial cells is preferably an integrin antagonist, selectinantagonist, Ig superfamily cell adhesion molecule antagonist, or CD44antagonist can preferably be tested and/or identified by applying such apotential antagonist to the flow chamber assay as described herein, inparticular in Example 2, whereby an antagonist preferably increases themean T cell-rolling velocity±SD (μm/sec) on HBMEC in the presence of +10ng/ml AMG 103 by about 30, 40, 50, 60, 70, 80, 90, 100% or even more, asdescribed above.

A compound acting on the T cell side as described above is characterized

-   (a) to be capable of binding to a T-cell adhesion molecule,-   (b) to be capable of blocking the binding site of a T-cell adhesion    molecule, and/or-   (c) to inhibit or reduce the expression of a T-cell adhesion    molecule.

A “T cell adhesion molecule” is a molecule that is present on thesurface of a T cell and that functions or has a role in the adhesion ofa T cell to other cells such as endothelial cells, the latter beingpreferred in the context of the present invention. Typically, a T celladhesion molecule interacts with an adhesion molecule of an endothelialcell. The adhesion molecule of an endothelial cells may thus be regardedas ligand for the T cell adhesion molecule. An interaction between a Tcell adhesion molecule and a ligand present on the surface of anendothelial cell usually takes place between a binding site of a T celladhesion molecule (i.e., a ligand binding site) and its designatedligand, i.e., an adhesion molecule of an endothelial cell.

A T cell adhesion molecule as described herein has preferably animmunoglobulin superfamily domain. Such an adhesion molecule ispreferably an integrin that has preferably an RGD-binding domain, suchas alpha4-integrin, alphaL-beta2-integerin, alphaL-integrin,beta7-integrin; a selectin such as L-selectin, or CD44. T cells useintegrins and/or selectins and/or CD44 to migrate in and out of vesselsand/or lymph nodes to then migrate, e.g. into other tissues, theleptomeningeal or perivascular space. CD44 mainly acts as receptor ofhyaluronic acid which is present on the surface of endothelial cellswhich line up vessels.

A compound of the present invention is in one embodiment characterizedto be capable of binding to a T cell adhesion molecule. As such, acompound binds and thus preferably sheds the T cell adhesion moleculesuch that the T cell adhesion molecule is diminished, preferably nolonger able to interact with an endothelial adhesion molecule. As aconsequence of a compound binding to a T cell adhesion molecule, a Tcell is at least diminished, preferably no longer able to interact withan endothelial adhesion molecule. A preferred compound that binds to a Tcell adhesion molecule is a molecule that is capable of binding toanother molecule, such as a lipocalin mutein, or an antibody, preferablya monoclonal antibody. Preferably, the binding molecule can bespecifically targeted to the T cell adhesion molecule. Since an antibodyfulfills that criterion, a preferred compound that binds to a T celladhesion molecule is an antibody, preferably a monoclonal antibody, suchas Natalizumab, Efalizumab, or Etrolizumab.

A compound of the present invention is additionally or alternativelycharacterized to be capable of blocking the binding site of a T-celladhesion molecule. “Blocking” means to prevent the binding site of a Tcell adhesion molecule from interacting with its ligand, preferably withthe binding site of its ligand, on the endothelial cell. As aconsequence of a compound blocking the binding site of a T cell adhesionmolecule, a T cell is at least diminished, preferably no longer able tointeract with an endothelial adhesion molecule. Non-limiting examples ofbinding sites of a T cell adhesion molecule are binding sites forICAM-1, ICAM-2, ICAM-3, VCAM-1, MadCAM, GlyCAM, CD31 (PECAM-1), CD62P(P-selectin), CD62E (E-selectin), CD62L, fibrinogen, and chondroitin.

Preferred compounds that block the binding site of a T-cell adhesionmolecule are soluble fragments of cell adhesion molecules of endothelialcells, said soluble fragments are preferably modified so as to bind tothe binding site of a T cell adhesion molecule without causing aphysiological effect such as the transduction of a signal to the T cell.

A preferred compound that blocks the binding site of a T cell adhesionmolecule is an antibody, preferably a monoclonal antibody that blocks anICAM-1, ICAM-2, ICAM-3, VCAM-1, MadCAM, GlyCAM, CD31, CD62P, CD62E,CD62L. fibrinogen, and/or chondroitin binding site on a T cell adhesionmolecule, e.g. on VLA-4 and/or LPAM-1, respectively. Such an antibody isNatalizumab which is thus a preferred compound applied in the presentinvention.Also preferred as a compound that is applied in the present invention isan antibody, preferably monoclonal antibody that blocks an ICAM-1binding site on a T cell adhesion molecule. Such an antibody isEfalizumab which is thus a preferred compound applied in the presentinvention.

Further preferred as a compound that is applied in the present inventionis an antibody, preferably monoclonal antibody that blocks a VCAM-1and/or MadCAM binding site on a T cell adhesion molecule. Such anantibody is Etrolizumab which is thus a preferred compound applied inthe present invention.

Another preferred compound is AJM300, a small molecule that blocksbinding sites of T cell adhesion molecules.

Another preferred compound is SAR 1118, a small molecule that blocks theICAM-1, ICAM-2 and/or ICAM-3 binding site on a T cell adhesion molecule.

Another further preferred compound is BOL-303225-A, a small moleculethat acts an antagonist of the integrins alphaLbeta2 and/or alphaMbeta2.

Another preferred compound is a chelator, preferably a chelator forbivalent cations, such as calcium. A preferred chelator isEthylenediaminetetraacetic acid (EDTA).

Hyaluronic acid (HA) or chondroitin sulfate are further preferredcompounds, since both are known to block the HA and E-selectin bindingsite on CD44, thereby blocking the interaction between CD44 on T cellsand endothelial cells.

A compound of the present invention is additionally or alternativelycharacterized by inhibiting or reducing the expression of a T-celladhesion molecule. Such a compound may act on the expression includingtranscription and/or translation of genes encoding T cell adhesionmolecules, such as suppressing gene expression, interfering withtranscription, splicing, or translation. This could, for example, beachieved by RNA interference by means and methods known in the art.However, other compounds are known which reduce the expression of a Tcell adhesion molecule, such as Minocycline which reduces LFA-1expression, (acetyl-)salicyclic acid which reduces L-selectinexpression, Astilbin or Flavonoids which reduce CD44 expression (seealso Table 1 herein). These compounds are preferred compounds that areapplied in the context of the present invention, with Minocycline beingmore preferred

The compound acting on the endothelial cell side is characterized

-   (a) to be capable of binding to an endothelial adhesion molecule,-   (b) to be capable of blocking the binding site of an endothelial    adhesion molecule, and/or-   (c) to inhibit or reduce the expression of an endothelial adhesion    molecule,

An “endothelial cell adhesion molecule” is a molecule that is present onthe surface of an endothelial cell and that functions or has a role inthe adhesion of endothelial cells to other cells such as leukocytes, inparticular T cells or monocytes. Typically, an adhesion molecule of anendothelial cell interacts with a T cell adhesion molecule. The adhesionmolecule of a T cell may thus be regarded as ligand for the endothelialcell adhesion molecule. An interaction between an endothelial celladhesion molecule and a ligand present on the surface of a T cellusually takes place between a binding site of an endothelial celladhesion molecule (i.e., a ligand binding site) and its designatedligand, i.e., an adhesion molecule of an T cell.

An endothelial cell adhesion molecule as described herein has preferablyan immunoglobulin superfamily domain. Such an adhesion molecule ispreferably an integrin that has preferably an RGD-binding domain, suchas ICAM-1, ICAM-2, ICAM-3, VCAM-1, GlycAM-1, or MadCAM. Via integrinsand/or selectins endothelial cells communicate and interact with, e.g. Tcells such that T cells can finally extravasate and migrate as describedabove.

A compound of the present invention is in one embodiment characterizedto be capable of binding to an endothelial adhesion molecule. As such, acompound binds and thus preferably sheds the endothelial adhesionmolecule such that adhesion of T cells in particular is diminished,preferably endothelial cells are no longer able to interact with a Tcell adhesion molecule. As a consequence of a compound binding to anendothelial cell adhesion molecule, an endothelial cell is at leastdiminished, preferably no longer able to interact with a T cell adhesionmolecule. A preferred compound that binds to an endothelial adhesionmolecule is a molecule that is capable of binding to another molecule,such as a lipocalin mutein, or an antibody, preferably a monoclonalantibody. Preferably, the binding molecule can be specifically targetedto the endothelial adhesion molecule. Since an antibody fulfills thatcriterion, a preferred compound that binds to an endothelial adhesionmolecule is an antibody, preferably a monoclonal antibody, such asPF-00547659.

A compound of the present invention is additionally or alternativelycharacterized to be capable of blocking the binding site of anendothelial adhesion molecule. “Blocking” means to prevent the bindingsite of an endothelial adhesion molecule from interacting with itsligand, preferably with the binding site of its ligand, on the T cell.As a consequence of a compound blocking the binding site of anendothelial adhesion molecule, an endothelial cell is at leastdiminished, preferably no longer able to interact with a T cell adhesionmolecule. Non-limiting examples of binding sites of an endothelial celladhesion molecule are binding sites for alpha4-integrins,alphaL-beta2-integrins, alphaL-integrins, beta7-integrins.

Preferred compounds that block the binding site of an endothelial celladhesion molecule are soluble fragments of cell adhesion molecules of Tcells, said soluble fragments are preferably modified so as to bind tothe binding site of an endothelial cell adhesion molecule withoutcausing a physiological effect such as the transduction of a signal tothe T cell.

A preferred compound that blocks the binding site of an endothelial celladhesion molecule is an antibody, preferably a monoclonal antibody thatblocks an alpha4-integrins, alphaL-beta2-integrins, alphaL-integrins,beta7-integrins binding site on an endothelial cell adhesion molecule.

Also preferred as a compound that is applied in the present invention isan antibody, preferably monoclonal antibody that binds an endothelialcell adhesion molecule, such as ICAM-1, ICAM-2, ICAM-3, VCAM-1,GlycAM-1, MadCAM, or PECAM-1.

Further preferred as a compound that is applied in the present inventionis an antibody, preferably monoclonal antibody that binds P-selection(CD62P). Such an antibody is Inclacumab.

Another preferred compound is thrombin, preferably at low pMconcentrations, that blocks binding sites of endothelial cell adhesionmolecules.

A further preferred compound is pentosanpolysulfate (PPS) that blocksthe PSGL-1 binding site on an endothelial cell adhesion molecule.

A compound of the present invention is additionally or alternativelycharacterized by inhibiting or reducing the expression of an endothelialcell adhesion molecule. Such a compound may act on the expressionincluding transcription and/or translation of genes encoding endothelialcell adhesion molecules, such as suppressing gene expression,interfering with transcription, splicing, or translation. This could,for example, be achieved by RNA interference by means and methods knownin the art. However, other compounds are known which reduce theexpression of an endothelial cell adhesion molecule, such asrosuvastatin, a small molecule, that reduces expression of VCAM-1.

The present invention also relates to a method of identifying a compoundfor use in a method of prophylaxis and/or amelioration and/or treatmentof clinical adverse events caused by therapy which comprisesre-directing of T-cells against target cells in a patient, comprising:

-   -   (a) contacting said compound with a mammalian T-cell, a        mammalian endothelial cell, a T-cell adhesion molecule and/or an        endothelial adhesion molecule; and    -   (b) evaluating whether said compound:        -   (i) decreases or inhibits the binding of mammalian T-cells            to mammalian endothelial cells;        -   (ii) is capable of binding to a T-cell adhesion molecule,        -   (iii) is capable of blocking the binding site of a T-cell            adhesion molecule,        -   (iv) inhibits or reduces the expression of a T-cell adhesion            molecule,        -   (v) is capable of binding to an endothelial adhesion            molecule,        -   (vi) is capable of blocking the binding site of an            endothelial adhesion molecule, and/or        -   (vii) inhibits or reduces the expression of an endothelial            adhesion molecule.

It is intended to use for example the flow system disclosed herein forthat purpose. A non-exclusive list of possible other in vitro assayswhich may be used for identifying a compound for use in a method ofprophylaxis and/or amelioration and/or treatment of clinical adverseevents caused by therapy which comprises re-directing of T-cells againsttarget cells in a patient, are depicted below:

-   -   Transmigration assays (Rohnelt, Hoch et al. 1997, Ding, Xiong et        al. 2000)    -   Adhesion assays with immobilized adhesion molecules (Gerli,        Gresele et al. 2001, Valignat, Theodoly et al. 2013)    -   Adhesion assays with endothelial cells and T cells under static        conditions (Mobley and Shimizu 2001)    -   Interference of such compounds with adhesion molecules on T        cells in flow cytometric assays (Bucolo, Maltese et al. 2008)

Bucolo, C., A. Maltese, F. Maugeri, K. W. Ward, M. Baiula, A. Sparta andS. Spampinato (2008). “New coumarin-based anti-inflammatory drug:putative antagonist of the integrins alphaLbeta2 and alphaMbeta2.” JPharm Pharmacol 60(11): 1473-1479.

Ding, Z., K. Xiong and T. B. Issekutz (2000). “Regulation ofchemokine-induced transendothelial migration of T lymphocytes byendothelial activation: differential effects on naive and memory Tcells.” J Leukoc Biol 67(6): 825-833.

Gerli, R., P. Gresele, O. Bistoni, C. Paolucci, L. Lanfrancone, S.Fiorucci, C. Muscat and V. Costantini (2001). “Salicylates inhibit Tcell adhesion on endothelium under nonstatic conditions: induction ofL-selectin shedding by a tyrosine kinase-dependent mechanism.” J Immunol166(2): 832-840.

Mobley, J. L. and Y. Shimizu (2001). “Measurement of cellular adhesionunder static conditions.” Curr Protoc Immunol Chapter 7: Unit 7 28.

Rohnelt, R. K., G. Hoch, Y. Reiss and B. Engelhardt (1997).“Immunosurveillance modelled in vitro: naive and memory T cellsspontaneously migrate across unstimulated microvascular endothelium.”Int Immunol 9(3): 435-450.

Valignat, M. P., O. Theodoly, A. Gucciardi, N. Hogg and A. C. Lellouch(2013). “T lymphocytes orient against the direction of fluid flow duringLFA-1-mediated migration.” Biophys J 104(2): 322-331.

As exemplified in Table 1 herein, many target molecules both onleukocytes and endothelial cells as well as corresponding interferingcompounds have been identified, are currently developed or have alreadybeen approved for use in humans. Therefore, it is envisaged to encompassin the present invention any existing or future compound regardless ofits mode of action that demonstrates anti-adhesive effects onleukocytes, and more preferably T cells in the flow system as definedherein for the administration prior to, concurrently with and/orsubsequently to the treatment of a patient with a therapy comprisingre-directing of T cells against target cells e.g. through T celltransduction with a CAR or T cell recruitment via a compound comprisinga CD3-specific binding domain, preferably Blinatumomab, for theprophylaxis or amelioration of CNS AEs caused by said (respective)therapy comprising re-directing of T cells against target cells.

Thus, in another embodiment the present invention relates to anycompound that demonstrates anti-adhesive effects on leukocyte rollingand adhesion in the described flow system. Addition of said compound orany combination of such compounds to the flow system is expected toneutralize leukocyte adhesion induced by re-directing of T cells againsttarget cells e.g. through T cell transduction with a CAR or T cellrecruitment via a compound comprising a CD3-specific binding domain,preferably Blinatumomab-induced T cell-adhesion to blood vessel-liningendothelial cells, e.g. T cell-rolling velocity is reverted to levelscomparable to those without Blinatumomab addition to the flow system.The present invention further relates to a combination of one or more ofsaid compounds with the treatment of a patient with a therapy comprisingre-directing of T cells against target cells e.g. through T celltransduction with a CAR or T cell recruitment via a compound comprisinga CD3-specific binding domain prior to, concurrently with and/orsubsequently to said treatment for the prophylaxis or amelioration ofCNS AEs caused by said (respective) therapy comprising re-directing of Tcells against target cells. In other words, any combinational treatmentof (a) compound(s) with anti-adhesive properties and a therapycomprising re-directing of T cells against target cells e.g. through Tcell transduction with a CAR or T cell recruitment via a compoundcomprising a CD3-specific binding domain is envisaged to be encompassedby the present invention. Yet in other words, it is envisaged as arequirement to administer any of said compounds or any combinationthereof prior to, concurrently with and/or subsequently to the treatmentof a patient with a therapy comprising re-directing of T cells againsttarget cells e.g. through T cell transduction with a CAR or T cellrecruitment via a compound comprising a CD3-specific binding domain forthe prophylaxis or amelioration of CNS AEs caused by said (respective)therapy comprising re-directing of T cells against target cells.

In the context of the present invention, “anti-leukocyte adhesion” isdefined as any prophylactic and/or interventional measure, method and/orprocedure to prevent, minimize, reduce, influence, mitigate or modifyleukocyte rolling on, binding to, adhesion to, transmigration through orinteraction with endothelial cells, preferably blood vessel-liningendothelial cells. Additionally, compounds included in such measures,methods and/or procedures, or exerting any of the above listed effectson leukocytes are hereby defined as “compounds with anti-leukocyteadhesion effects” or “compounds with anti-adhesive properties” or“compounds of the invention” or “compound which decreases or inhibitsthe binding of mammalian T-cells to mammalian endothelial cells”.

According to the current hypothesis leukocyte adhesion to blood vesselendothelium is a necessary prerequisite for the induction of severeadverse effects such as CNS AEs requiring treatment discontinuation.Thus, a possible mitigation approach for AEs, such as CNS AEs, is, inthe context of the present invention, anti-leukocyte adhesion aiming atreducing endothelial adherence of re-directed T cells and optionallyalso other leukocytes (i.e. T lymphocytes, natural killer (NK) cells,neutrophil granulocytes and monocytes, which are the most prominenttypes of extravasating mammalian cells) following start of infusion andstepwise dose increases of the respective medicament, i.e. the therapywhich comprises re-directing of T-cells against target cells in apatient, for example Blinatumomab, which therapy is the cause forendothelial cell-activation and leukocyte extravasation into the CNS(see the discussion herein and the results derived from the appendedexamples). The term “mammalian T-cells” comprises or consists of“re-directed T-cells”, as these re-directed T-cells are causative forthe AEs as explained herein. Thus, in a preferred embodiment of thepresent invention, said “mammalian T-cell” is a “re-directed mammalianT-cell”. Re-directing of T cells comprises that T cells are equippedwith an antigen receptor specificity recognizing a target cells whichtypically differs from the T cells' clonotypic natural antigen receptorspecificity. This can e.g. be achieved by T cell engaging bi- ormulti-functional antibodies or antibody derivatives which comprise aspecific binding domain that is capable of specifically binding to aT-cell receptor, preferably to CD3. It is also possible (and explicitlyenvisaged within the context of the present invention) that suchre-directed T-cells are produced by transduction of T cells withchimeric antigen receptors (CARs), for example CARs recognizing CD19(see exemplarily Knochenderfer et al., Nature Reviews 2013; ClinicalOncology; “Treating B-cell cancer with T cells expressing anti-CD19chimeric antigen receptors”). A “re-directed T-cell” thus includesT-cells that have been contacted with bi- or multi-functional antibodiesor antibody derivatives which comprise a specific binding domain that iscapable of specifically binding to a T-cell receptor (preferably to CD3)or a T-cell that has been genetically engineered to express a chimericantigen receptor (i.e. a T-cell CAR—see WO2007/131092 which is includedherein by reference).

In a particularly preferred embodiment said re-directed T-cell is ahuman T-cell that has been contacted with (is bound by) Blinatumomab(AMG103).

A “therapy which comprises re-directing of T-cells against target cells”is to be understood as a therapy, such as a medicament, which ischaracterized by the appearance and/or existence of “re-directedT-cells”, i.e. the therapy either comprises or consists of re-directedT-cells as such, for example genetically engineered T-cell having achimeric antigen receptor CAR (optionally formulated as a pharmaceuticalcomposition) and/or the re-directed T-cells appear in the course of thetherapy exemplified by a medicament which comprises a CD3-specificbinding domain as defined herein, preferably a CD3-specific bindingdomain together with a binding domain which is specific for B-cells,more preferably a CD3-specific binding domain together with a bindingdomain which is specific for a CD-marker that can be found on B-celllymphoma such as CD19, CD22, CD20 or CD79a, CD19 being preferred. In amore preferred embodiment, said therapy which comprises re-directing ofT-cells against target cells is a therapy with a bispecific CD3 X CD19antibody and in a most preferred embodiment said therapy which comprisesre-directing of T-cells against target cells is a therapy withBlinatumomab.

It is also envisaged that said therapy with Blinatumomab encompasses theadministration of 5 to 10 μg/m²/day or higher doses, such as 15, 45 or60 μg/m²/day. The above mentioned CD-3 specific binding domains areexplained in great detail herein elsewhere.

Chimeric antigen receptors (CARs) are fusion proteins comprising antigenrecognition moieties and T cell-activation domains. For the treatment ofB cell-malignancies, CD19 CARs consisting of a CD19-specific bindingdomain linked to, e.g. CD3zeta have been described in clinical studiesfor B CLL (Porter et al. N Engl J. Med. 2011; 365:725-33) and B ALL(Grupp et al. N Engl J. Med. 2013). As observed with the infusion of aCD19×CD3 bispecific single-chain antibody, adoptive transfer of CD19CAR-transduced T cells into patients led to rapid and sustainederadication of normal and malignant B cells. Common adverse eventsassociated with CD19 CAR T cell-therapy included cytokine releasesyndrome and lymphopenia, but cases of CNS AEs were also reported. Thus,interference with adhesion and transmigration of CD19 CAR T cellsto/through blood vessel-lining endothelium also is a useful approach forthe prophylaxis and/or amelioration of CD 19 CAR T cell-induced CNS AEs.Of note, it is envisaged that treatment with CAR T cells targeting otherB cell-specific antigens (e.g. CD20) would also benefit fromco-medication with compounds with anti-adhesive properties for theprophylaxis and/or amelioration of CNS AEs caused by such CAR T cells.

The “chimeric antigen receptor (CAR)” as used herein comprises a bindingdomain which is specific for B-cells, preferably specific for aCD-marker that can be found on B-cell lymphoma such as CD19, CD22, CD20or CD79a, CD19 being preferred. T-cells that have been geneticallyengineered to express a chimeric antigen receptor CAR (a T-cell CAR) areexemplified in WO2007/131092. It is meanwhile known that also a therapycomprising T-cell CARs triggers clinical adverse events, and inparticular CNS AE.

The term “target cells” is not specifically limited and relatespreferably to cancer target cells (in particular cancer cells thatexpress a suitable target which makes them attackable). B-lymphoma cellsare more preferred, CD19 positive B-cells (B-lymphoma cells) being mostpreferred.

The term “mammalian” includes but is not limited to mouse, rat, dog,horse, camel, primates, etc., primates being preferred and human beingsbeing most preferred.

The mammalian “endothelial cells” can be isolated from large vessels orcapillaries. The term “endothelial cells” thereby includes freshlyisolated endothelial cells (for example HUVECs), commercially availableendothelial cells from different manufacturers (e.g. PromoCell) andendothelial cell lines, although endothelial cell lines are lesspreferred. Human endothelial cells are preferred. Human Umbilical VeinEndothelial Cells (HUVEC) and Human Brain Microvascular EndothelialCells (HBMEC) are particularly preferred, HBMECs being most preferred.

Adverse events can be classified in five grades in accordance with theCommon Terminology Criteria for Adverse Events (CTCAE). Grade 1 relatesto mild AEs, Grade 2 to moderate AEs, Grade 3 to severe AEs, Grade 4 tolife-threatening or disabling AEs, while Grade 5 means death related toAEs. All these AEs are contemplated within the framework of the presentinvention and included by the term “clinical adverse events” or “adverseeffects” or related terms used herein.

The term “clinical adverse events” used herein caused by therapy whichcomprises re-directing of T-cells against target cells in a patientcomprises in particular neurological adverse events. Said neurologicaladverse event, which sometimes is also denoted as “neurological symptom”or “neurological adverse effect” or “central nervous system adverseevent (CNS AE)”, includes but is not limited to conditions of a humanpatient such as all forms of pain, headache, muscleweakness/incoordination, balance disorder, speech disorder/impairment,sensual disturbance/abnormalities, dizziness, ataxia, apraxia, tremor,aphasia, dysphasia, confusion, disorientation, hallucination, cerebellarsymptoms, encephalopathy, seizure, (grand mal) convulsion. Specifically,neurological symptoms observed during treatment with a therapycomprising re-directing of T cells against target cells e.g. through Tcell transduction with a CAR or T cell recruitment via a compoundcomprising a CD3-specific binding domain include for example confusionand disorientation. “Confusion” as used herein refers to loss oforientation which is the ability to place oneself correctly in the worldby time, location, and personal identity, and often memory which is theability to correctly recall previous events or learn new material. Thepatients usually have difficulties to concentrate and thinking is notonly blurred and unclear but often significantly slowed down. Patientswith neurological symptoms also suffer from loss of memory. Frequently,confusion leads to loss of the ability to recognize people and/orplaces, or to tell time and date. Feelings of disorientation are commonin confusion, and the decision-making ability is impaired. Neurologicalsymptoms further comprise blurred speech and/or word-findingdifficulties. This disorder may impair the expression and understandingof language as well as reading and writing. Additionally, vertigo anddizziness may accompany neurological symptoms in some patients.

The term “clinical adverse events” is preferably characterized by (butis not limited to) one or more of (i) cognitive disorder comprisingdisorientation/confusion and/or word finding problems/aphasia, (ii)seizure, (iii) cerebellar symptoms partly observed as an optionalprodromal phase of (i) or (ii) comprising kinetic tremor, ataxia,dysarthria and handwriting problems. Further neurological adverse eventare apraxia and hallucination. Word finding problems are preferred inthe context of the present invention. Of particular clinicalsignificance are clinical adverse effects which cause discontinuation oftreatment with therapy which comprises re-directing of T-cells againsttarget cells in a patient (preferably therapy with Blinatumomab or otherCD3-binding drugs or CARs), because the treated patient thus cannotfully benefit from the treatment.

The “patient” is a mammalian patient, preferably a primate, mostpreferably a human being.

In a preferred embodiment the patient is suspected/assumed to compriseor already comprises malignant CD19-positive B cells. In the latter casesaid patient has already been diagnosed to comprise such cells. Themalignant CD19-positive B cells are present in a patient developingand/or suffering from lymphoma and/or leukemia.

The present invention also relates to a nucleic acid which encodes achimeric antigen receptor (CAR) for use in a method of re-directing ofT-cells against target cells in a patient, wherein said patient issubject to therapy comprising the “compound which decreases or inhibitsthe binding of mammalian T-cells to mammalian endothelial cells”. Anucleic acid sequence thereby includes, although not being limitedthereto, vectors etc., which will allow the expression of the desiredCARs in T-cells (see for example WO2007/131092 which is included hereinby reference).

For the avoidance of any doubt, it is hereby stressed that thedisclosure of the present invention including all definitions etc. isfully applicable to all embodiments that form part of the presentinvention (i.e. are linked with the gist of the invention and thereforefall into the context of the present invention), irrespective of whetherthese embodiments are drafted as compounds for use embodiments or methodof treatment embodiments or compound embodiments, kit embodiments,composition embodiments, use embodiments, method embodiments etc. Thus,all definitions and embodiments can be used and apply to all embodimentsdisclosed herein. The present invention thus also relates to a methodfor prophylaxis, amelioration and/or treatment of clinical adverseevents caused by therapy which comprises re-directing of T-cells againsttarget cells in a patient, said method comprising administering atherapeutically effective amount of a compound which decreases orinhibits the binding of mammalian T-cells to mammalian endothelialcells. The term “therapeutically effective amount” is meant to refer toan amount of the compound which decreases or inhibits the binding ofmammalian T-cells to mammalian endothelial cells that provides atreatment, amelioration or prophylaxis of clinical adverse events causedby therapy which comprises re-directing of T-cells against target cellsin a patient (i.e. that provides “therapeutic efficacy”).

In the following it is intended to further illustrate the etiology ofcentral nervous system adverse events (CNS AEs) caused by a CD3-specificbinding domain and the rationale for employing anti-leukocyte adhesionas prophylaxis and amelioration of such CNS AEs.

CNS AEs requiring treatment discontinuation are currently best explainedby the hypothesis of a multi-step pathomechanism initiating transientmeningeal or perivascular neuroinflammation.

Start of infusion with Blinatumomab and any stepwise dose increaseinduce rapid adhesion of peripheral blood T cells, along with otherperipheral blood leukocytes such as NK cells and monocytes, to bloodvessel-lining endothelial cells. This process occurs irrespective of thepresence of circulating (i.e. peripheral blood) target cells, i.e.normal and/or malignant CD19-positive B cells. Moreover, Tcell-redistribution as a result of T cell-adhesion to blood vesselendothelium seems to be at least partially independent of theBlinatumomab dose as it was observed even at the lowest dose level of0.5 μg/m²/day tested. Accordingly, T cell-redistribution as a result ofa transient increase in T cell-adhesiveness appears to be triggered bythe exposure change per se at start of Blinatumomab administration andduring dose increases rather than by the higher absolute exposure at ahigher dose. The endothelial adhesion of peripheral blood leukocytes isvery likely to also take place at blood vessels of the CNS whereespecially meningeal microvessels have been suggested as initial entrysite for inflammatory cells from peripheral blood and as a potentialstarting point for neuroinflammatory phenomena.

As described in more detail in herein, three key findings support thisassumption: (1) the redistribution of circulating T cells and otherleukocytes after start of infusion or stepwise dose increase in anypatient involving a rapid decrease of blood cell-counts already within45 minutes followed by a recovery of blood cell-counts within a fewdays; (2) the transient increase of adhesiveness of circulating T cellsto blood vessel-lining endothelial cells as measured by increasedbinding of soluble ICAM-1-F_(c) fusion proteins to LFA-1 on T cells; and(3) the transient increase of angiopoietin-2 (Ang-2) in peripheralblood, which represents a specific marker for the activation ofendothelial cells (Fiedler and Augustin. Trends Immunol. 2006;27:552-8).

Following adhesion of leukocytes to meningeal microvessels andpostcapillary brain venules, part of these cells are thought toextravasate into leptomeningeal and perivascular spaces, respectively(FIGS. 2A and B). Even in the absence of profound T cell-extravasation,leukocyte adhesion to blood vessel-lining endothelial cells per se mayinduce leakage of CNS blood vessels and neurological symptoms such asseizures (Fabene et al. Nat. Med. 2008; 14:1377-83).

Pentosanpolysulfate (PPS; C₁₄H₂₆O₂₁S₄), also known as PentosanPolysulphate, Xylan Hydrogen Sulfate, Xylan Polysulfate, is asemi-synthetically produced heparin-like macromolecular carbohydratederivative, which chemically and structurally resemblesglucosaminoglycans. It is a white odorless powder, slightly hygroscopicand soluble in water to 50% at pH 6. It has a molecular weight of 4000to 6000 Dalton.

PPS is, for example, sold under the name Elmiron® by Ortho-McNeilPharmaceutical, Inc. and is thus far the only oral medication approvedby the U.S. FDA for the treatment of interstitial cystitis, also knownas painful bladder syndrome. For treatment of this condition, PPS isadministered orally, however, it can alternatively be administeredintravenously.

The term “pentosanpolysulfate (or PPS)” encompasses semi-syntheticallyproduced heparin-like macromolecular carbohydrate derivatives.

In the sense of the present invention, pentosanpolysulfate is a mixtureof linear polymers of β1→4-linked xylose, usually sulfated at the 2- and3-positions and occasionally substituted at the 2-position with4-O-methyl-α-D-glucuronic acid-2,3-O-sulfate. Accordingly, PPS may alsobe designated as β1→4-D-Xylan-2,3-bis(hydrogen sulfate).

By way of example, a semi-synthetically produced heparin-likemacromolecular carbohydrate derivative such as in particular PPS is, forexample, producible (obtainable) as follows: its polysaccharidebackbone, xylan is, for example, extracted from the bark of the beechtree or other plant sources and is then treated with sulfating agentssuch as chlorosulfonic acid or sulfuryl chloride and acid. Aftersulfation, PPS is usually treated with sodium hydroxide to yield thesodium salt which is a preferred salt of the present invention.Processes for the production of a semi-synthetically producedheparin-like macromolecular carbohydrate derivative such as inparticular PPS are, for example, disclosed in U.S. Pat. No. 2,689,848 orUS 2010/0105889.

In the context of the present invention PPS is preferably administeredorally to a patient, even more preferably intravenously. Typical dosesare 100, 150, 200 or 300 mg, administered 1-3 times per day, with amaximum amount of 600 mg/day. Typically, the daily dose is between 100and 600 mg such as 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or600 mg or even more. For example, 100 mg PPS may be administered 3-6times. Similarly, 200 mg PPS may be administered 2-3 times.Alternatively, 300 mg PPS may be administered 2 times. Alternatively,PPS in an amount, for example, between 100 and 600 mg such as 100, 150,200, 250, 300, 350, 400, 450, 500, 550 or 600 mg may be administeredover 24 hours via infusion, for example, by using a perfusor. In thelatter case, a bolus injection of PPS in an amount, for example, of 100,200 or 300 mg may precede a therapy comprising re-directing of T cellsagainst target cells in a patient, followed by administration of PPS of100, 200 or 300 mg/day over 24, 48 or 72 hours.

PPS may be administered prior to (e.g. prophylactically as a bolusinjection), concurrently with or subsequently to the administration of atherapy comprising re-directing of T cells against target cells in apatient as described herein. Advantageously, PPS may be administered toa patient when the therapy comprising re-directing of T cells againsttarget cells in a patient will be repeated or increased (e.g. at startof infusion or any dose steps). A particular preferred PPS that wasadministered to a patient mentioned in the present invention ispentosanpolysulfate SP54® commonly known and available in the market(e.g. from bene Arzneimittel GmbH).

Apart from PPS, other clinically available compounds with knownanti-adhesive properties are Minocycline and Natalizumab as describedherein. While P-selectin mediates the first step (i.e. rolling) ofleukocyte adhesion to endothelial cells, the subsequent steps areintegrin-mediated. Particularly, the interactions of LFA-1 and VLA-4 onT cells with ICAM-1 and VCAM-1 on endothelial cells, respectively, playa prominent role during this second phase of leukocyte adhesion toendothelial cells. As such, since LFA-1, VLA-4 and ICAM-1 belong to thefamily of integrins and IgG superfamily, integrin antagonists and IgGsuperfamily antagonists are preferred compounds of the presentinvention.

As small-molecule inhibitors of LFA-1, tetracyclines are available forclinical use in humans. Within the tetracycline family, Minocycline isthe best-characterized inhibitor of LFA-1 (Nikodemova et al. JNeuroimmunol. 2010; 219:33-7). Minocycline is the most lipophilic amongall tetracyclines and has an almost 100% bioavailability after oraladministration. It also has the longest in vivo half-life ofapproximately 24 hours which is the prerequisite for an uninterruptedserum exposure over a prolonged period of time. Moreover, Minocyclinepenetrates best into the CNS which makes it particularly suitable forthe treatment of CNS disorders. In clinical trials, Minocycline wasshown to counteract neuroinflammation in multiple sclerosis (Zhang etal. Can J Neurol Sci. 2008; 35:185-91) and to improve the neurologicaloutcome in patients with acute ischemic stroke (Lampl et al. Neurology.2007; 69:1404-10). In a recent dose-finding study on Minocycline asneuroprotective agent in stroke patients, daily doses of up to 10 mg/kgiv equivalent to 700 mg per os for several days were found to be safeand well-tolerated (Fagan et al. Stroke. 2010; 41:2283-7).Mechanistically, Minocycline both down-regulates expression of LFA-1 onT cells and acts as a chelator for cations such as Ca²⁺ and Mg²⁺ whichare required for firm binding of LFA-1 to ICAM-1. Thus, as said above,Minocyclin is a preferred compound of the present invention.

Natalizumab is an antibody approved for the treatment of multiplesclerosis. It binds to VLA-4 on T cells thereby blocking its interactionwith VCAM-1 on endothelial cells. In consequence, T cell-adhesion andextravasation, especially into the brain, are diminished. Hence,Natalizumab is another preferred compound of the present invention.

A non-exclusive list of other compounds with proposed anti-adhesiveproperties that are or might become available for clinical use in humansis provided in Table 1. Each of these compounds is a preferred compoundof the present invention, with Minocyclin and Natalizumab being morepreferred.

In connection with the present invention a “CD3-specific binding domain”sometimes also denoted herein as “CD3 binding domain” characterizes abinding domain which comprises a framework/framework region and an“antigen-binding site” or an “antigen-interaction site” which is able tospecifically interact with a CD3 antigen. Said binding/interaction isalso understood to define a “specific recognition”. The term“specifically interact/interacting” means in accordance with thisinvention that the binding domain is capable of binding to an epitope ofthe CD3 antigen, preferably the CD3epsilon antigen, and more preferablythe human CD3epsilon antigen.

As used herein, “CD3” denotes a molecule expressed as part of the Tcell-receptor complex and has the meaning as typically ascribed to it inthe prior art. In humans, it encompasses in individual or independentlycombined form all known CD3 subunits, for example CD3epsilon, CD3delta,CD3gamma and CD3zeta. The human CD3epsilon antigen is indicated inGenBank Accession No. NM_000733.

The term “framework (region)” includes a scaffold for antigen-bindingsites. For example, such a scaffold could be provided by protein A, inparticular the Z-domain thereof (affibodies), ImmE7 (immunity proteins),BPTI/APPI (Kunitz domains), Ras-binding protein AF-6 (PDZ-domains),charybdotoxin (Scorpion toxin), CTLA-4, Min-23 (knottins), lipocalins(anticalins), neokarzinostatin, a fibronectin domain, an ankyrinconsensus repeat domain or thioredoxin (Skerra. Curr Opin Biotechnol.2007; 18:295-304; Hosse et al. Protein Sci. 2006; 15:14-27; Nicaise etal. Protein Sci. 2004; 13:1882-91; Nygren and Uhlén. Curr Opin StructBiol. 1997; 7:463-9).

In the context of the present invention a preferred framework is theart-recognized portions of an antibody variable region that existbetween the more divergent (i.e. hypervariable) complementaritydetermining regions (CDRs) within the variable region of an antibody.Such framework regions are typically referred to as frameworks 1 through4 (FR1, FR2, FR3, and FR4) and provide scaffolds for the presentation ofthe six CDRs (three from the heavy chain and three from the light chain)in three dimensional space, to form an antigen-binding surface.

A preferred example of a CD3-specific binding domain in line with thepresent invention is an antibody. The CD3-specific binding domain may bea monoclonal or polyclonal antibody or derived from a monoclonal orpolyclonal antibody. The term “antibody” comprises derivatives orfunctional fragments thereof which still retain the binding specificity.Techniques for the production of antibodies are well known in the artand described, e.g. in Harlow and Lane “Antibodies, A LaboratoryManual”, Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane“Using Antibodies: A Laboratory Manual”, Cold Spring Harbor LaboratoryPress, 1999. The term “antibody” also comprises immunoglobulins (Ig's)of different classes (i.e. IgA, IgG, IgM, IgD and IgE) and subclasses(such as IgG1, IgG2, etc.). The definition of the term “antibody” alsoincludes embodiments such as chimeric, single-chain, de-immunized andhumanized antibodies, as well as antibody fragments, like, inter alia,Fab fragments. Antibody fragments or derivatives further compriseF(ab′)₂, Fv, scFv fragments or single domain antibodies, single variabledomain antibodies or immunoglobulin single variable domain comprisingmerely one variable domain, which might be VH or VL, that specificallybind to an antigen or epitope independently of other V regions ordomains; see, for example, Harlow and Lane (1988) and (1999), citedabove. Such immunoglobulin single variable domain encompasses not onlyan isolated antibody single variable domain polypeptide, but also largerpolypeptides that comprise one or more monomers of an antibody singlevariable domain polypeptide sequence.

Bispecific antibody formats are preferred; however, other multispecificantibody formats (trispecific, tetrabodies, etc.) are not excluded. Itis preferred that said CD3 binding domain is contained in or iscomprised by a bispecific single chain antibody. Said bispecific singlechain antibody further comprises in another preferred embodiment of thepresent invention a binding domain which is specific for B-cells,preferably specific for a CD-marker that can be found on B-cell lymphomasuch as CD19, CD22, CD20 or CD79a, CD19 being preferred. In aparticularly preferred embodiment, said bispecific single chain antibodyis a CD19×CD3 or CD20×CD3 bispecific single chain antibody. In an evenmore preferred embodiment, said CD19×CD3 bispecific single chainantibody is Blinatumomab (MT103/AMG103). In a further preferredembodiment of the present invention said CD19×CD3 bispecificsingle-chain antibodies comprise a first binding domain capable ofbinding to an epitope of human CD3epsilon and a second binding domaincapable of binding to human CD 19. The human CD-Antigens are easilyderivable from publicly available databases. The human CD19 antigen isfor example indicated in GenBank Accession No. AAA69966.

All the specific CD19×CD3 bispecific single-chain antibodies disclosedtherein, including their variants, fragments, equivalents, etc. areparticularly preferred CD19×CD3 bispecific single-chain antibodies ofthe present invention.

As used herein, a “CD19×CD3 bispecific single-chain antibody” denotes asingle polypeptide chain comprising two binding domains. Such bispecificsingle-chain antibodies are preferred in the context of themethods/dosage regimen of the present invention. Each binding domaincomprises at least one variable region from an antibody heavy chain (“VHor H region”), wherein the VH region of the first binding domainspecifically binds to CD3epsilon and the VH region of the second bindingdomain specifically binds to CD19. The two binding domains areoptionally linked to one another by a short polypeptide spacer. Anon-limiting example for a polypeptide spacer is Gly-Gly-Gly-Gly-Ser(G-G-G-G-S) and repeats thereof. Each binding domain may additionallycomprise one variable region from an antibody light chain (“VL or Lregion”), the VH region and VL region within each of the first andsecond binding domains being linked to one another via a polypeptidelinker, for example of the type disclosed and claimed in EP 623679 B 1,but in any case long enough to allow the VH region and VL region of thefirst binding domain and the VH region and VL region of the secondbinding domain to pair with one another such that, together, they areable to specifically bind to the respective first and second bindingdomain. Such CD19×CD3 bispecific single-chain antibodies are describedin great detail in WO 99/54440 and WO 2004/106381.

Preferably, the bispecific single-chain antibody applied in themethods/dosage regimen of the present invention has the domainarrangement (a) VL(CD19)-VH(CD19)-VH (CD3)-VL(CD3). However, it is alsoenvisaged that the methods of the invention can be carried out withCD19×CD3 bispecific single-chain antibodies of other domainarrangements, such as

-   (b) VH(CD19)-VL(CD19)-VH(CD3)-VL(CD3),-   (c) VL(CD19)-VH(CD19)-VL(CD3)-VH(CD3),-   (d) VH(CD19)-VL(CD19)-VL(CD3)-VH(CD3),-   (e) VL(CD3)-VH(CD3)-VH(CD19)-VL(CD19),-   (f) VH(CD3)-VL(CD3)-VH(CD19)-VL(CD19),-   (g) VL(CD3)-VH(CD3)-VL(CD19)-VH(CD19), or-   (h) VH(CD3)-VL(CD3)-VL(CD19)-VH(CD19).

A preferred CD19×CD3 bispecific single-chain antibody applied in themethods of the present invention comprises the

-   (a) anti-CD3 CDRs of the heavy chain shown as CD3 CDR-H1 in SEQ ID    NO: 11 (RYTMH), more preferably in SEQ ID NO: 11 (GYTFTRYTMH), CD3    CDR-H2 in SEQ ID NO: 12 (YINPSRGYTNYNQKFKD) and CD3 CDR-H3 in SEQ ID    NO: 13 (YYDDHYCLDY); and/or-   (b) anti-CD3 CDRs of the light chain shown as CD3 CDR-L1 in SEQ ID    NO: 14 (RASSSVSYMN), CD3 CDR-L2 in SEQ ID NO: 15 (DTSKVAS) and CD3    CDR-L3 in SEQ ID NO: 16 (QQWSSNPLT); and/or-   (c) anti-CD19 CDRs of the heavy chain shown as CD19 CDR-H1 in SEQ ID    NO: 17 (SYWMN), more preferably in SEQ ID NO: 17 (GYAFSSYWMN), CD19    CDR-H2 in SEQ ID NO: 18 (QIWPGDGDTNYNGKFKG) and CD19 CDR-H3 in SEQ    ID NO: 19 (RETTTVGRYYYAMDY); and/or-   (d) anti-CD19 CDRs of the light chain shown as CD19 CDR-L1 in SEQ ID    NO: 20 (KASQSVDYDGDSYLN), CD19 CDR-L2 in SEQ ID NO: 21 (DASNLVS) and    CD19 CDR-L3 in SEQ ID NO: 22 (QQSTEDPWT).

It is more preferred that the CD19×CD3 bispecific single-chain antibodyapplied in the methods of the present invention comprises the CD3 CDRsof the heavy and light chain. Even more preferably, the CD19×CD3bispecific single-chain antibody applied in the methods of the presentinvention comprises the CD3 CDRs of the heavy and light chain as well asthe CD19 CDRs of the heavy and light chain.

The CDRs referred to herein are in accordance with the Kabat numberingsystem. The Kabat numbering scheme is a widely adopted standard fornumbering the residues of an antibody in a consistent manner (Kabat etal., Sequences of Proteins of Immunological Interest, 1991).

Alternatively, it is preferred that the CD19×CD3 bispecific single-chainantibody applied in the methods of the present invention comprises the

-   (a) CD19 variable heavy chain shown in SEQ ID NO: 3 (nucleotide    sequence is shown in SEQ ID NO: 4); and/or-   (b) CD19 variable light chain shown in SEQ ID NO: 5 (nucleotide    sequence is shown in SEQ ID NO: 6); and/or-   (c) CD3 variable heavy chain shown in SEQ ID NO: 7 (nucleotide    sequence is shown in SEQ ID NO: 8); and/or-   (d) CD3 variable light chain shown in SEQ ID NO: 9 (nucleotide    sequence is shown in SEQ ID NO: 10).

More preferably, the CD19×CD3 bispecific single-chain antibody appliedin the methods of the present invention comprises the CD19 variableheavy and light chain and/or the CD3 variable heavy and light chain.Even more preferably, the CD19×CD3 bispecific single-chain antibodyapplied in the methods of the present invention comprises the CD19variable heavy and light chain as well as the CD3 variable heavy andlight chain.

In another alternative, it is also preferred that the CD19×CD3bispecific single-chain antibody comprises an amino acid sequenceselected from the group consisting of

-   (a) an amino acid sequence as depicted in SEQ ID NO: 1;-   (b) an amino acid sequence encoded by a nucleic acid sequence as    shown in SEQ ID NO: 2;-   (c) an amino acid sequence encoded by a nucleic acid sequence having    at least 70%, 80%, 90%, 95% or 99% identity to a nucleic acid    sequence of (b), wherein said amino acid sequence is capable of    specifically binding to CD3 and CD 19; and-   (d) an amino acid sequence encoded by a nucleic acid sequence which    is degenerate as a result of the genetic code to a nucleotide    sequence of (b), wherein said amino acid sequence is capable of    specifically binding to CD3 and CD 19.

It is to be understood that the sequence identity is determined over theentire amino acid sequence. For sequence alignments, for example, theprograms Gap or BestFit can be used (Needleman and Wunsch. J Mol Biol.1970; 48:443-53; Smith and Waterman. Adv Appl Math. 1981; 2:482-9),which are contained in the GCG software package (Genetics ComputerGroup, 575 Science Drive, Madison, Wis., USA 53711). It is a routinemethod for those skilled in the art to determine and identify an aminoacid sequence having e.g. 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%sequence identity to the amino acid sequences of the CD19×CD3 bispecificsingle-chain antibodies described herein (preferably Blinatumomab). Forexample, according to Crick's Wobble hypothesis, the 5′ base on theanti-codon is not as spatially confined as the other two bases, andcould thus have non-standard base pairing. In other words: the thirdposition in a codon triplet may vary so that two triplets which differin this third position may encode the same amino acid residue. Saidhypothesis is well known to the person skilled in the art (see e.g.http://en.wikipedia.org/wiki/Wobble_Hypothesis; Crick. J Mol. Biol.1966; 19:548-55). It is furthermore a routine procedure for thoseskilled in the art to determine cytotoxic activity of such an amino acidsequence having e.g. 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequenceidentity to the nucleotide or amino acid sequences of the CD19×CD3bispecific single-chain antibodies described herein. Cytotoxic activityof the CD19×CD3 bispecific single-chain antibody or an antibodyconstruct having e.g. 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequenceidentity to the amino acid sequences of the CD19×CD3 bispecificsingle-chain antibodies described herein can be determined by methods asillustrated, e.g. in WO 99/54440.

It is particularly preferred, that said CD19×CD3 bispecific single-chainantibody has the amino acid sequence shown in SEQ ID NO: 1. Alsoparticularly preferred is the CD19×CD3 bispecific single-chain antibodyas described in WO 99/54440 as well as those CD19×CD3 bispecificsingle-chain antibodies described in WO 2004/106381. Blinatumomab (orAMG 103 or MT103) is most preferred.

The present invention further relates to a CD19×CD3 bispecificsingle-chain antibody and/or a method for

-   (a) treating malignant CD19-positive cells, preferably lymphocytes,    even more preferably B cells, in a human patient, and/or-   (b) administering a CD19×CD3 bispecific single-chain antibody to a    human patient;    wherein a compound with anti-adhesive properties is to be    administered prior to, concurrently with or subsequently to the    treatment of a human patient with said CD19×CD3 bispecific    single-chain antibody for the prophylaxis or amelioration of CNS AEs    caused by said CD19×CD3 bispecific single-chain antibody.    a compound which decreases or inhibits the binding of mammalian    T-cells to mammalian endothelial cells for use in a method of    prophylaxis and/or amelioration and/or treatment of clinical adverse    events caused by therapy which comprises re-directing of T-cells    against target cells in a patient

The administration of the therapy which comprises re-directing ofT-cells against target cells in a patient (preferably the CD3-specificbinding domain, more preferably Blinatumomab), of (a) compound(s) withanti-adhesive properties (compound which decreases or inhibits thebinding of mammalian T-cells to mammalian endothelial cells) and/or of(a) pharmaceutical composition(s) comprising either or a combination ofthe aforementioned components, is preferably an intravenousadministration. It may be administered as a bolus injection or bycontinuous (continual) intravenous (civ) infusion, with continuouslybeing preferred. A continuous administration refers to an administrationwhich essentially is without interruption. “Essentially withoutinterruption” includes a continuous administration which usually iswithout interrupted flow or spatial extension. By way of example, WO2007/068354 discloses a treatment regimen which is specifically includedherein by way of reference thereto. Other treatment regimens which areenvisaged in the context of the present invention are disclosed inPCT/EP2010/066207.

It is also envisaged that the patient is characterized by a B/T-cellratio of less than 1:5 (see PCT/EP2010/066207) and/or a B-cell number ofless than about 50 B-cells per μl peripheral blood. As disclosed ingreat detail in PCT/EP2010/066207, the administration of a CD3-specificbinding domain, in particular of a CD19×CD3 bispecific single-chainantibody to a patient is frequently accompanied by neurological symptomsif said patient is characterized by a B:T cell-ratio of less than 1:5.However, the herein disclosed prophylaxis or amelioration of theseneurological adverse effects caused by a CD3-specific binding domain byway of co-medication with (a) compound(s) having anti-adhesiveproperties (as defined in the present invention) is also applicable topatients who are characterized by a B:T cell-ratio equal to or of morethan 1:5 (see PCT/EP2010/066207).

The present invention also relates to a (pharmaceutical) kit or packagecomprising a “compound” (i.e. a compound which decreases or inhibits thebinding of mammalian T-cells to mammalian endothelial cells as definedherein) and/or a “therapy which comprises re-directing of T-cellsagainst target cells” (also defined herein, preferably a CD3-specificbinding domain and most preferred Blinatumomab), and instructions, alabel and/or an imprint indicating that said compound is to be employedfor use in a method of prophylaxis and/or amelioration and/or treatmentof clinical adverse events caused by therapy which comprisesre-directing of T-cells against target cells in a patient. Additionallyor alternatively, said instructions, label and/or imprint indicate thata therapy which comprises re-directing of T-cells against target cells,may cause AEs, in particular CNS AEs and that it is thereforerecommended, envisaged or necessary to mitigate these effects with acompound which decreases or inhibits the binding of mammalian T-cells tomammalian endothelial cells. The compounds and/or the therapy whichcomprises re-directing of T-cells against target cells, is/arepreferably packaged in one sealed kit or package. It is also envisagedthat this kit or package further comprises means to administer thecontent(s) to a patient, and/or buffers, vials, teflon bags or infusionbags which are normally used for the infusion of therapeutic agents.“Means” thereby includes one or more article(s) selected from the groupconsisting of a syringe, a hypodermic needle, a cannula, a catheter, aninfusion bag for intravenous administration, intravenous vehicles,vials, buffers, stabilizers, written instructions which aid the skilledperson to prepare the respective doses and infusions of the invention,etc.

The above mentioned (pharmaceutical) kit or package may also comprise anucleic acid which encodes a chimeric antigen receptor (CAR).

Tables

TABLE 1 Compounds with known or proposed anti-adhesive properties thatare or might become available for clinical use in humans. TargetCompound Mode of Action Reference Endothelial cell adhesion molecules(also denoted as targets on endothelial cells) P-selectin PPS, smallmolecule Blockage of PSGL-1 Höpfner et al. J binding site PharmPharmacol. 2003; 55: 697-706 Inclacumab, mAb Blockage of PSGL-1 Kling etal. Thromb binding site Res. 2013 ICAM-1 Alicaforsen enema Inhibition ofICAM-1 Van Deventer et al. (ISIS2303), siRNA expression AlimentPharmacol Ther. 2006; 23: 1415- 25 MAdCAM PF-00547659, mAb Blockage ofβ7- Pullen et al. Br J integrins binding site Pharmacol. 2009; 157:281-93 VCAM-1 Rosuvastatin, small Reduction of VCAM- Osaka et al. Biomedmolecule 1 expression Res Int. 2013 PAR-1, Thrombin (at low pMAnti-leukocyte Ku and Bae. Inflamm fibrinogen concentrations) adhesiveeffect, Res. 2013 maintenance of vascular barrier integrity T-celladhesion molecules (also denoted as targets on T cells) α₄-integrinsNatalizumab, mAb Blockage of VCAM- Haanstra et al. J (e.g. VLA-4, 1,MAdCAM, Immunol. 2013 LPAM-1) fibrinogen, chondroitin binding siteAJM300, small Blockage of ligand Thomas and molecule binding siteBaumgart. Inflammophar- macology. 2012; 20: 1-18 α_(L)β₂-integrinMinocycline Reduction of LFA-1 Nikodemova et al. J (LFA-1) expression,ion Neuroimmunol. 2010; chelator 219: 33-7 SAR 1118, small Blockage ofICAM-1, Rao et al. Invest molecule -2, -3 binding site Ophthalmol VisSci. 2010; 51: 5198-204 BOL-303225-A, Antagonist Bucolo et al. J Pharmsmall molecule Pharmacol. 2008; 60: 1473-9 α_(L)-integrins Efalizumab,mAb Blockage of ICAM-1 Koszik et al. J (e.g. LFA-1) binding siteDermatol Sci. 2010; 60: 159-66 β₇-integrins Etrolizumab, mAb Blockage ofVCAM-1 Stefanich et al. Br J and MAdCAM Pharmacol. 2011; binding site162: 1855-70 α₅β₁-integrin Natramune (PDS- Interference with Weeks etal. Med Sci (VLA-5) 2865), PureWay-C, α₅β₁-integrin- Monit. 2008; 14:ascorbic acid; small mediated adhesion BR279-85; Eylar et molecule al. PR Health Sci J. 1996; 15: 21-6 L-selectin (Acetyl-) salicylic Reductionof L- Gerli et al. J acid selectin expression Immunol. 2001; 166: 832-40Integrins Ethylenediaminetetra- Ion chelator Welzenbach et al. J (e.g.LFA-1, acetic acid (EDTA) Biol Chem. 2002; VLA-4) 277: 10590-8 CD44Hyaluronic acid Blockage of HA and Murai et al. Immunol (HA),chondroitin E-selectin binding site Lett. 2004; 93: 163- Sulfate,anti-CD44 70; Baaten et al. mAb Front Immunol. 2012; 3: 23 Astilbin,flavonoid Reduction of CD44 Yi et al. Int expression Immunopharmacol.2008; 8: 1467-74 CD162 mAb Blockage of P- Moore. Leuk (PSGL-1) selectinbinding site Lymphoma. 1998; 29: 1-15 Src-family PP2 (4-Amino-5-(4-Selective inhibitor of Feigelson et al. J Biol kinaseschlorophenyl)-7-(t- Src-family kinases Chem. 2001; 276: (e.g. Lck,butyl)pyrazolo[3,4- 13891-901 Fyn) d]pyrimidine) Targets on T cellsand/or endothelial cells Sialidase Sialidase inhibitors, Blockage ofFeng et al. J Leukoc e.g. castanospermine; desialylation of, e.g. Biol.2011; 90: 313- small molecule LFA-1, ICAM-1 21 Adhesion Recombinantligands Competitive blockage molecules of respective adhesion of naturalligand molecules binding siRNA, shRNA Reduction or inhibition ofexpression of respective adhesion molecules mAb: monoclonal antibody

TABLE 2 Characteristics of freshly isolated human T cells. Marker CD3CD4 CD8 CD11a CD49d CD162 CD69 CD25 HLA-DR % 93.37 ± 68.87 ± 25.10 ±99.93 ± 32.90 ± 99.87 ± 1.10 ± 49.60 ± 5.03 ± positive  9.41  4.02  4.50 0.06  2.04  0.23 0.52 13.85 2.15 cells ± SD

The expression of CD3, CD4, CD8, CD11a, CD49d, CD162 (PSGL-1), CD69,CD25, and HLA-DR on the surface of freshly isolated human T cells wasdetermined by flow cytometry. CD3-positive cells are expressed as meanpercentage±SD of all events of 3 independent measurements. CD4, CD8,CD11a, CD49d, CD162, CD69, CD25, and HLA-DR-positive cells are expressedas mean percentages±SD of CD3-positive cells of 3 independentmeasurements.

The compound of the present invention is preferably anon-glucocorticoidal compound, i.e. glucocorticoids are preferablyexcluded. The term “glucocorticoid” means compounds that bind,preferably specifically, to the glucocorticoid receptor. Said termincludes compound(s) selected from the group consisting of cortisone,cortisol (hydrocortisone), cloprednol, prednisone, prednisolone,methylprednisolone, deflazacort, fluocortolone, triamcinolone,dexamethasone, beatamethasone, cortivazol, paramethasone, and/orfluticasone, including pharmaceutically acceptable derivatives thereof.

EXAMPLES

The following examples illustrate the invention. These examples shouldnot be construed as to limit the scope of this invention. The examplesare included for purposes of illustration.

Example 1 Successful Mitigation of CNS AEs by Anti-leukocyte Adhesionwith PPS in Patients at High Risk of Discontinuation of BlinatumomabTreatment Due to CNS AEs

Dosing Regimen of Blinatumomab and PPS Co-medication.

In a phase 1 clinical study three patients were treated withBlinatumomab at an initial dose of 5 μg/m²/day for 1 week followed by adose escalation to 60 μg/m²/day for additional 3 to 7 weeks. ConcomitantPPS was administered as 100 mg bolus iv infusion at 3 h±30 min prior tostart of infusion and dose escalation of Blinatumomab followed byperfusion of 300 mg/day for 48 h after start of infusion and doseescalation of Blinatumomab.

Patient 109-036 (See Also FIG. 3E).

Patient 109-036, a male Caucasian, age 62, weight 96.8 kg, height 178cm, presented with Follicular Lymphoma grade I/II. Previous therapiesincluded CHOP (02/12-03/04), Dexa BEAM (03/05), Cyclophosphamid (03/08),and radio therapy followed by autologous stem cell transplantation(03/08). The patient achieved a complete response (CR) after 57 days ofciv infusion of Blinatumomab. Although this patient had a high risk ofdeveloping CNS AEs due to his low B:T cell-ratio (seePCT/EP2010/066207), no treatment discontinuation due to CNS AEs wasrequired.

Patient 109-040 (See Also FIG. 3F).

Patient 109-040, a male Caucasian, age 51, weight 94.0 kg, height 180cm, presented with Lymphoplasmocytic Lymphoma (Morbus Waldenström).Previous therapies included CVP (04/12-05/03), Leukeran (05/10-05/12),Rituximab (06/05-06/07), R-CHOP (06/07-06/10), Dexa BEAM (06/10), BEAM(06/12), and radio therapy followed by autologous stem celltransplantation (06/12). Additional prior therapies comprisedRituximab+Bendamustine (09/04-09/12) and Rituximab (09/04-09/12). Thepatient achieved a stable disease (SD) after 30 days of civ infusion ofBlinatumomab. Although this patient had a high risk of developing CNSAEs due to his low B:T cell-ratio (see PCT/EP2010/066207), no treatmentdiscontinuation due to CNS AEs was required.

Patient 109-042 (See Also FIG. 3D).

Patient 109-042, a female Caucasian, age 49, weight 92.0 kg, height 169cm, presented with Follicular Lymphoma grade I/II. Previous therapiesincluded R-CHOP (09/11-10/02) and Rituximab (10/03-10/04). The patientachieved a complete response (CR) after 56 days of civ infusion ofBlinatumomab. Although this patient had a high risk of developing CNSAEs due to her low B:T cell-ratio (see PCT/EP2010/066207), no treatmentdiscontinuation due to CNS AEs was required.

Example 2 In Vitro Flow Chamber System Mimicking T Cell/endothelialCell-Interaction Triggered by Blinatumomab or Other CD3-Specific BindingDrugs

Cultivation of Endothelial Cells.

Human brain microvascular endothelial cells (HBMEC) (#1000, SciencellResearch Laboratories) or human umbilical vein endothelial cells (HUVEC)(#C-12200, Promocell) were used as endothelial cell-model. CryopreservedHBMEC at passage 1 (>5×10⁵ cells/ml) were first cultivated infibronectin-coated Nunclon-treated 75 cm² cell culture flasks (#156499,Nunc) at 37° C. and 5% CO₂ in a Heraeus Cytoperm 2 (Thermo Scientific)according to the manufacturer's instructions. Sub-cultivation of HBMECwas done with the Detach Kit (#C-41200, Promocell) consisting ofHEPES-buffered balanced salt solution (#C-40000, Promocell),trypsin-EDTA solution (0.04%/0.03%) (#C-41000, Promocell) and trypsinneutralization solution (TNS, #C-41100, Promocell). Briefly, the mediumwas aspirated from the HBMEC-layer and cells were washed with 2 ml ofHEPES-buffered balanced salt solution. Addition of 2 ml of trypsin-EDTAsolution for 1-5 min at room temperature led to detachment of HBMEC fromthe flask bottom. Inactivation of trypsin-EDTA solution was performed byaddition of 2 ml of trypsin neutralization solution to the cellsuspension. Cells were centrifuged at 300 g for 4 min in a HeraeusMegafuge 40 (Thermo Scientific) and seeded at a cell density of 5×10⁵cells per gelatin-coated 75 cm² cell culture flask (#L7230, Biochrom).HBMEC for rolling and adhesion experiments were cultivated in RPMI 1640medium (#FG1215, Biochrom) supplemented with Nu-Serum IV (10%) (#355505,BD Biosciences), dialyzed FBS (10%) (#SZ0115, Biochrom), MEM-vitamins(1%) (#K0373, Biochrom), L-glutamine (1%) (#K0283, Biochrom),sodium-pyruvate (1%) (#L0473, Biochrom), heparin (10 U/ml) (#L6510,Biochrom), and epidermal cell growth factor ECGS (30 μg/ml) (#02-102,Millipore). Cultivation of HUVEC was done in endothelial cell growthmedium (#C-22010, Promocell) supplemented with dialyzed FBS (10%)(#SZ0115, Biochrom) using gelatin-coated 75 cm² cell culture flasks.Sub-cultivation of HUVEC was performed with the Detach Kit (#C-40000,Promocell) and cells were seeded at a cell density of 4.0-7.5×10⁵ cellsper 75 cm² cell culture flask.

Isolation of Human Peripheral Blood Mononuclear Cells (PBMC) and T Cellsfrom Blood.

Isolation of human PBMC from blood was performed by modified densitygradient centrifugation as described elsewhere. Briefly, 15-20 ml offreshly drawn heparinized blood was transferred in Biocoll (#L6115,Biochrom)-containing Leukosep tubes (#227290, Greiner bio-one) andcentrifuged in a Hettich Rotanta 460 RS Type 5606 (HettichLaborapparate) at 1066 g for 15 min. After removal of the plasmafraction the PBMC containing phase was transferred in a new tube andwashed twice with FACS-buffer (D-PBS #L1820, 5% FBS #SO115, Biochrom).Erythrocyte lysis was performed with lysis buffer (8.29 g/l NH₄Cl, 1.00g/l KHCO₃, 0.037 g/l EDTA) for 5 min at room temperature. For subsequentpurification of untouched T cells the human Pan T cell-isolation Kit II(#130-091-156, Miltenyi Biotec) was used according to the manufacturer'sinstructions.

Characterization of Isolated Human T Cells by Flow Cytometry.

Isolated T cells were characterized by flow cytometry for surfaceexpression of adhesion molecules and activation markers. FACS stainingand washing procedures were performed at 4° C. in cold FACS-buffer(D-PBS with 5% FBS). To analyze surface-exposed adhesion molecules,3×10⁵ T cells were stained in 100 μl of FACS-buffer with anti-CD11a-APC(1:10) (#550852, BD Biosciences), anti-CD49d-PE (1:10) (#560972, BDBiosciences), and anti-CD162-PerCP-eFluor710 (1:20) (#46-1629,eBioscience). Possible activation of T cells was monitored by staining3×10⁵ T cells with anti-CD69-PE (1:40) (#555531, BD Biosciences),anti-CD25-APC (1:40) (#340907, BD Biosciences), and anti-HLA-DR-FITC(1:40) (#555811, BD Biosciences). Furthermore, anti-CD3-V450 (1:40)(#560365, BD Biosciences), anti-CD4-APC-Cy7 (1:40) (#341115, BDBiosciences), and anti-CD8-V500 (1:40) (#560774, BD Biosciences) wereincluded in both staining to characterize T cell-subpopulations.Separate staining of 3×10⁵ T cells with DAPI (1 μg/ml) (#A1001.0010,Applichem) allowed for monitoring of cell viability. Staining for 30 minwas followed by washing of cells twice with FACS-buffer. Stained sampleswere subjected to FACS analysis on a FACSCanto II instrument (BDBiosciences) and statistical analysis was performed using the FACSDivasoftware (BD Biosciences). 10,000 events were recorded and CD4-, CD8-,CD11a-, CD49d-, CD162-, CD69-, CD25-, and HLA-DR-positive cells wereexpressed as percentage of CD3-positive cells.

Cultivation of Endothelial Cells Under Flow Conditions with the IbidiPump System.

The ibidi Pump System (#10902, ibidi) was used for cultivation ofendothelial cells under flow conditions as well as for performingadhesion and rolling assays of human T cells on endothelial cells. Thesystem consists of the ibidi Pump (#10905, ibidi) and the ibidi FluidicUnit (#10903, ibidi) which work together to generate a unidirectionalflow of medium in an attached slide with a defined channel height. Thesystem is controlled by the ibidi Pump Control software (#10908,notebook+software, ibidi). For cultivation of HBMEC or HUVEC under flowconditions, HBMEC or HUVEC were seeded at a cell density of 2.5×10⁶cells/ml into μ-slide I^(0.4) Luer Collagen IV (#80172, ibidi) orμ-slide I^(0.4) Luer ibiTreat (#80176, ibidi), respectively according tothe manufacturer's instructions and cultivated with a perfusion set“yellow/green” (#10964, ibidi) for 48 h under wall shear stress of 5 or10 dyn/cm², respectively. For the cultivation of more than one μ-slideI^(0.4) Luer with a single fluidic unit, up to four μ-slides wereconnected to each other with the Serial Connectors for μ-slides (#10830,ibidi).

For the additional pre-incubation of HBMEC with pentosanpolysulfateSP54® (100 mg/ml injection solution, bene Arzneimittel GmbH), PPS wasadded to HBMEC cell culture medium (200 μg/ml) 24 h prior to anyexperiments. Histamine pre-stimulation of HBMEC was done prior torolling and adhesion experiments with 10⁻⁵ M histamine (#H7125-1G,Sigma-Aldrich) in HBMEC cell culture medium for 30 min at 37° C.

Incubation of Human T Cells with Compounds Mediating Anti-LeukocyteAdhesion.

For further interference with AMG 103-mediated adhesion effects, T cellswere pre-incubated with compounds potentially mediating anti-leukocyteadhesion prior to rolling and adhesion experiments. Therefore, Tysabri(Natalizumab, 20 mg/ml solution, Elan Pharma International Ltd.) orMinocin (Minocycline, 100 mg/vial, Triax Pharmaceuticals) were added tothe T cell-suspension and incubated at 37° C.

Interaction of Human T Cells with HBMEC or HUVEC Under Flow Conditions:Assay Setup for Rolling and Adhesion Experiments.

Experiments under defined hydrodynamic flow conditions were performed byusing freshly isolated human T cells and flow-cultivated HBMEC or HUVECas described above, and were analyzed with a microscopic systemconsisting of the inverse microscope Ti-E (#MEA53100, Nikon), thedigital camera Orca Flash 2.8 (#C-11440-10C, Hamamatsu), theNIS-Elements AR software versions 3.22.00 and 4.10.03 (#MQS31200 and#MQS31100, Nikon), the ibidi Pump System (#10902, ibidi), the cellculture incubator Galaxy 14S (#C014S-120-0000, Eppendorf), the HeatingSystem 8 (#10925, ibidi), and the CO₂ gas incubation unit I (#10920,ibidi). The microscope Ti-E was equipped with a TI-ND6-PFS Perfect FocusSystem (#MEP59390, Nikon) allowing for the correction of parfocalvariances. Rolling and adhesion events were monitored with a 10×objective (CFI PlanFluor DL-10 X phase, #MRH20101, Nikon). The HeatingSystem 8 and the CO₂ gas incubation unit I were started at least 3 hbefore starting any experiment, pre-warming the stage top incubator to37° C. and 5% CO₂. RPMI 1640 medium (#FG1215, Biochrom) was alsopre-warmed at 37° C. and 5% CO₂. For rolling and adhesion experimentsthe μ-slide I^(0.4) Collagen IV with flow-cultivated HBMEC or theμ-slide I^(0.4) ibiTreat with flow-cultivated HUVEC was disconnectedfrom the perfusion set, rinsed with pre-warmed RPMI 1640 medium with PPS(200 μg/ml) or without PPS and placed in the μ-slide inlet of the stagetop incubator under the microscope. 6×10⁶ freshly isolated human T cellswere centrifuged in PC V-tubes (#347759, Nunc) at 300 g for 4 min. Tcells were resuspended in RPMI 1640 medium with PPS (200 μg/ml) orwithout PPS to a final T cell-density of 1×10⁶ cells/ml. These cellswere either used directly for rolling experiments with or withoutaddition of AMG 103 (10 ng/ml) to the cell suspension, or furtherpre-incubated in PC V-tubes at 37° C. for 45 min in the presence orabsence of AMG 103. Such prepared T cell-suspension was filled in aperfusion set “white” (#10963, ibidi) which was connected to both afluidic unit and the μ-slide. Two different setup for rolling andadhesion experiments of T cells on HBMEC or HUVEC at a shear stress of 1dyn/cm² were applied:

-   1. Rolling of T cells on endothelial cells for 45 sec (short-term    condition)-   2. Rolling of T cells on endothelial cells for 45-120 min (long-term    condition, with fluidic unit being placed in the cell culture    incubator)

Interaction of Human T Cells with HBMEC or HUVEC Under Flow Conditions:Data Acquisition and Analysis.

T cell-interactions with endothelial cells were monitored by imageacquisition with digital camera Orca Flash 2.8 which in turn wascontrolled by NIS-Elements software 3.22.00. Time-lapse 45 secacquisition (without delay) was performed for individual time pointsresulting in up to 45 frames/sec at a resolution of 1920×1440. Suchrecorded image sequences of 45 sec duration were subjected to theautomated tracking module of NIS-Elements AR 4.10.03 or subjected tomanual tracking with NIS-Elements AR 3.22.00. Generated tracking datawere exported to Microsoft Excel and further analyzed by applyingfilters on parameters such as average heading, average speed and pathlength for each tracked object. Subsequently, mean rollingvelocity±standard deviation of all filtered cells was calculated. Whenusing manual tracking, 10-40 T cells were selected, tracked manually andthe mean rolling velocity±standard deviation was determined.

Immunofluorescence Staining of HBMEC or HUVEC.

After rolling and adhesion of T cells on HBMEC or HUVEC under flowconditions, endothelial cells were fixed using 150 μl of 4%paraformaldehyde solution (#P-6148, Sigma-Aldrich) for 30 min at 4° C.μ-slides were rinsed with 150 μl of D-PBS and subjected toimmunofluorescence staining. HBMEC were first blocked with 150 μl ofavidin blocking reagent (#PHA-70871, reagent 1, Dianova) for 10 min atroom temperature, washed with 150 μl of D-PBS followed by incubationwith 150 μl of biotin blocking reagent (#PHA-70871, reagent 2, Dianova).All following staining procedures were performed in 150 μl of D-PBS with5% FBS at room temperature in the dark, and washing steps were done in150 μl of D-PBS. μ-slides were incubated with 5 μg/ml polyclonal rabbitanti-human VCAM-1 (#106777, abcam) for 1 h. After washing, 20 μg/ml goatanti-rabbit IgG-DyLight350 was added for 1 h followed by washing andincubation with 15 μg/ml mouse anti-human P-selectin IgG1 (#BBA30, R&DSystems) for 2 h. After washing, goat anti-mouse IgG-Alexa488 (1:100)(#A10680, Invitrogen) was added for 1 h. ICAM-1 staining was done with10 μg/ml polyclonal rabbit anti-human ICAM-1 biotin (#AB7815, abcam) for1 h, followed by washing and incubation with streptavidin-Cy3 (1:100)(#016-160-084, Dianova) for 1 h. Cell surface staining of ICAM-1 andP-selectin on PFA-fixed HUVEC was performed as described for HBMEC.Finally, HBMEC or HUVEC were subjected to microscopic analysis usingUV-light and a CFI Plan Apochromat DM 20 x Lambda objective (#MRD30205,Nikon) with a PH-2 phase module (#MEH41200, Nikon). VCAM-1 staining wasmonitored with a CFL EPI-FL Filter Block UV-2A (#MBE41200, Nikon),P-selectin staining with a CFL EPI-FL Filter Block GFP-B (#MBE44740,Nikon), and ICAM-1 staining with an EPI-FL Filter Block Cy3 (#MXU96213,Nikon). Image acquisition was performed with the NIS-Elements software.

Statistical Data Analysis.

Depending on how many groups of data were compared, an unpaired t-testor a one-way ANOVA combined with a Tukey post-test were used to analyzestatistical significance in Prism (GraphPad Software). A P-value<0.05was regarded as statistically significant.

T cells pre-incubated with 10 ng/ml of Blinatumomab (+AMG 103) for 45min showed a significantly reduced mean rolling velocity±SD of 237±45μm/sec on HUVEC in short-term conditions when compared to the negativecontrol (−AMG 103; 283±82 μm/sec). Simultaneously, pre-incubation with10 ng/ml of Blinatumomab increased the number of T cells firmly adheringto HUVEC by 2.6-fold. Additionally, HUVEC-activation was observed asshown by increased surface expression of adhesion molecules ICAM-1 andP-selectin when corresponding T cells were pre-incubated with 10 ng/mlof Blinatumomab compared to basal expression levels on HUVEC that onlyhad contact to untouched T cells. These findings demonstrated increasedinteraction and firm adhesion of Blinatumomab-incubated T cells with/toHUVEC thereby in turn activating and increasing adhesiveness of thesecells (FIG. 5).

When using HBMEC, mean T cell-rolling velocity±SD was significantlyreduced to 209±40 μm/sec in the presence of 10 ng/ml of Blinatumomab(+AMG 103) in long-term conditions compared to the negative control(−AMG 103; 323±78 μm/sec). This reduction was observed 45 min afteraddition of Blinatumomab to the flow system, while no decrease of Tcell-rolling velocity was detectable immediately after addition ofBlinatumomab (0 min). HBMEC having interacted withBlinatumomab-stimulated T cells showed increased surface expression ofadhesion molecules ICAM-1, P-selectin and VCAM-1 after T cell-rollingcompared to basal expression levels on HUVEC that only had contact tountouched T cells. These observations demonstrated Blinatumomab-inducedincreased T cell-interaction with HBMEC and simultaneous increasedHBMEC-adhesiveness which timely (i.e. after 45 min) correlated with timecourses of T cell-redistribution in clinical studies (FIG. 6).

The influence of PPS on Blinatumomab-induced reduction of mean Tcell-rolling velocity as observed 45 min after addition of 10 ng/ml ofBlinatumomab to the flow system was evaluated in long-term conditions.While AMG 103 significantly reduced mean T cell-rolling velocity±SD onHBMEC from 430±92 μm/sec (−AMG 103) to 281±96 μm/sec (+AMG 103), furtheraddition of PPS to the flow system reverted this reduction to a mean Tcell-rolling velocity±SD of 483±157 μm/sec as also observed in theabsence of AMG 103 (i.e. −AMG 103, +PPS; 442±156 μm/sec). Cell surfaceexpression of P-selectin on HBMEC was increased when Blinatumomab wasadded to the flow system, whereas further addition of (i.e.pre-incubation of HBMEC with) PPS led to a lesser increase. Thus,prevention of T cell-interaction with HBMEC by PPS also diminished Tcell-mediated increase of HBMEC-activation and adhesiveness (FIG. 7).

As described in detail above for PPS in combination with histaminepre-stimulated HBMEC, the flow system was also used to analyze theinterference of PPS with AMG 103-induced T cell-interactions withnon-stimulated HBMEC.

The influence of PPS on Blinatumomab-induced reduction of mean Tcell-rolling velocity was evaluated under long-term conditions. WhileAMG 103 significantly reduced mean T cell-rolling velocity±SD on HBMECfrom 399±153 μm/sec (−AMG 103) to 263±66 μm/sec (+AMG 103) at 40 minafter addition of 10 ng/ml Blinatumomab to the flow system, furtheraddition of PPS to the experiment reverted this reduction to a mean Tcell-rolling velocity±SD of 465±116 μm/sec as also observed in theabsence of AMG 103 (i.e., −AMG 103, +PPS; 514±159 μm/sec). Thus, PPSprevented the Blinatumomab-induced increase of T cell-interactions withendothelial cells (FIG. 8).

As described in detail above for PPS in combination with histaminepre-stimulated HBMEC, the flow system was also used to analyze theinterference of natalizumab with AMG 103-induced T cell-interactionswith non-stimulated HBMEC. To this end, freshly isolated T cells wereincubated with 1 μg/ml natalizumab in RPMI 1640 medium for 10 min at 37°C. prior to rolling and adhesion experiments.

The influence of natalizumab on Blinatumomab-induced reduction of mean Tcell-rolling velocity was evaluated under long-term conditions. WhileAMG 103 significantly reduced mean T cell-rolling velocity±SD on HBMECfrom 482±149 μm/sec (−AMG 103) to 359±102 μm/sec (+AMG 103) at 40 minafter addition of 10 ng/ml Blinatumomab to the flow system, furtheraddition of natalizumab to the experiment reverted this reduction to amean T cell-rolling velocity±SD of 444±110 μm/sec as also observed inthe absence of AMG 103 (i.e., −AMG 103, +natalizumab; 445±81 μm/sec).Thus, natalizumab prevented the Blinatumomab-induced increase of Tcell-interactions with endothelial cells (FIG. 9).

As described in detail above for PPS in combination with histaminepre-stimulated HBMEC, the flow system was also used to analyze theinterference of minocycline with AMG 103-induced T cell-interactionswith non-stimulated HUVEC. To this end, freshly isolated T cells wereincubated with 50 μg/ml minocycline in Dulbecco's PBS for 2 h at 37° C.prior to rolling and adhesion experiments.

The influence of minocycline on Blinatumomab-induced reduction of mean Tcell-rolling velocity was evaluated under long-term conditions. WhileAMG 103 significantly reduced mean T cell-rolling velocity±SD on HUVECfrom 169±μm/sec (−AMG 103) to 127±41 μm/sec (+AMG 103) at 40 min afteraddition of 10 ng/ml Blinatumomab to the flow system, further additionof minocycline to the experiment reverted this reduction to a mean Tcell-rolling velocity±SD of 217±92 μm/sec as also observed in theabsence of AMG 103 (i.e., −AMG 103, +minocycline; 233±29 μm/sec) (FIG.10).

In yet another experiment, the influence of minocycline onBlinatumomab-induced increase of absolute numbers of T cells firmlyadhering to HUVEC was evaluated under long-term conditions. The numberof adhering T cells at 40 min after addition of 10 ng/ml Blinatumomab tothe flow system was significantly increased (2.1-fold) in the presenceof Blinatumomab (+AMG 103) compared to T cells alone (−AMG 103). Furtheraddition of minocycline to the experiment (+AMG 103, +minocycline)reverted the increased number of adhering T cells to a comparable oreven lower level as observed in the absence of Blinatumomab (−AMG 103;±minocycline) (FIG. 11).

Thus, minocycline prevented the Blinatumomab-induced increase of Tcell-interactions with endothelial cells.

As described in detail above for PPS in combination with histaminepre-stimulated HBMEC, the flow system was also used to analyze theinterference of an anti-ICAM-1 antibody with AMG 103-induced Tcell-interactions with non-stimulated HBMEC. To this end, HBMEC wereincubated with 10 μg/ml mouse anti-human ICAM-1 antibody (#MAB2146Z,Millipore) for 30 min at 37° C. prior to rolling and adhesionexperiments.

The influence of the anti-ICAM-1 antibody on Blinatumomab-inducedreduction of mean T cell-rolling velocity was evaluated under long-termconditions. While AMG 103 significantly reduced mean T cell-rollingvelocity±SD on HBMEC from 407±90 μm/sec (−AMG 103) to 335±μm/sec (+AMG103) at 30 min after addition of 10 ng/ml Blinatumomab to the flowsystem, further addition of the anti-ICAM-1 antibody to the experimentreverted this reduction to a mean T cell-rolling velocity±SD of 416±97μm/sec as also observed in the absence of AMG 103 (i.e., −AMG 103,+anti-ICAM-1 Ab; 378±64 μm/sec). Thus, the anti-ICAM-1 antibodyprevented the Blinatumomab-induced increase of T cell-interactions withendothelial cells (FIG. 12).

As described in detail above for PPS in combination with histaminepre-stimulated HBMEC, the flow system was also used to analyze theinterference of an anti-P-selectin antibody with AMG 103-induced Tcell-interactions with non-stimulated HBMEC. To this end, HBMEC wereincubated with 10 μg/ml mouse anti-human P-selectin antibody (#MAB2154,Millipore) for 30 min at 37° C. prior to rolling and adhesionexperiments.

The influence of the anti-P-selectin antibody on Blinatumomab-inducedreduction of mean T cell-rolling velocity was evaluated under long-termconditions. While AMG 103 significantly reduced mean T cell-rollingvelocity±SD on HBMEC from 449±90 μm/sec (−AMG 103) to 370±55 μm/sec(+AMG 103) at 40 min after addition of 10 ng/ml Blinatumomab to the flowsystem, further addition of the anti-P-selectin antibody to theexperiment reverted this reduction to a mean T cell-rolling velocity±SDof 440±95 μm/sec as also observed in the absence of AMG 103 (i.e., −AMG103, +anti-P-selectin Ab; 494±105 μm/sec). Thus, the anti-P-selectinantibody prevented the Blinatumomab-induced increase of Tcell-interactions with endothelial cells (FIG. 13).

As described in detail above for PPS in combination with histaminepre-stimulated HBMEC, the flow system was also used to analyze theinterference of an anti-CD11a antibody with AMG 103-induced Tcell-interactions with non-stimulated HBMEC. To this end, freshlyisolated T cells were incubated with 5 μg/ml mouse anti-human CD11aantibody (#217640, Calbiochem) in RPMI 1640 medium for 10 min at 37° C.prior to rolling and adhesion experiments.

The influence of the anti-CD11a antibody on Blinatumomab-inducedreduction of mean T cell-rolling velocity was evaluated under long-termconditions. While AMG 103 significantly reduced mean T cell-rollingvelocity±SD on HBMEC from 304±40 μm/sec (−AMG 103) to 214±μm/sec (+AMG103) at 10 min after addition of 10 ng/ml Blinatumomab to the flowsystem, further addition of the anti-CD11a antibody to the experimentreverted this reduction to a mean T cell-rolling velocity±SD of 261±56μm/sec as also observed in the absence of AMG 103 (i.e., −AMG 103,+anti-CD11a Ab; 278±48 μm/sec). Thus, the anti-CD11a antibody preventedthe Blinatumomab-induced increase of T cell-interactions withendothelial cells (FIG. 14).

As described in detail above for PPS in combination with histaminepre-stimulated HBMEC, the flow system was also used to analyze theinterference of an anti-CD162 antibody with AMG 103-induced Tcell-interactions with non-stimulated HBMEC. To this end, freshlyisolated T cells were incubated with 10 μg/ml mouse anti-human CD 162antibody (#ab78188, abcam) in RPMI 1640 medium for 10 min at 37° C.prior to rolling and adhesion experiments. Alternatively, instead ofincubating T cells with the anti-CD162 antibody, T cells were incubatedwith 10 μg/ml mouse isotype control antibody (IgG1,κ; MOPC-21; #M5284,Sigma-Aldrich) in RPMI 1640 medium for 10 min at 37° C. prior to rollingand adhesion experiments.

The influence of the anti-CD162 antibody on Blinatumomab-inducedreduction of mean T cell-rolling velocity was evaluated under long-termconditions. While AMG 103 significantly reduced mean T cell-rollingvelocity±SD on HBMEC from 539±149 μm/sec (−AMG 103) to 428±89 μm/sec(+AMG 103) at 45 min after addition of 10 ng/ml Blinatumomab to the flowsystem, further addition of the anti-CD162 antibody to the experimentreverted this reduction to a mean T cell-rolling velocity±SD of 637±191μm/sec as also observed in the absence of AMG 103 (i.e., −AMG 103,+anti-CD162 Ab; 528±170 μm/sec). In contrast, addition of the isotypecontrol antibody in the presence of AMG 103 resulted in a mean Tcell-rolling velocity±SD of 258±65 μm/sec (+AMG 103, +mouse IgG1,κ) anddid not revert the AMG 103-induced reduction of mean T cell-rollingvelocity as observed for the anti-CD162 antibody. Thus, the anti-CD162antibody specifically prevented the Blinatumomab-induced increase of Tcell-interactions with endothelial cells by selectively interfering withthe binding of CD162 to P-selectin. This prevention could not be inducedby the unspecific mouse isotype control antibody (FIG. 15).

As described in detail above for PPS in combination with histaminepre-stimulated HBMEC, the flow system was also used to analyze theinterference of recombinant P-selectin with AMG 103-induced Tcell-interactions with non-stimulated HBMEC. To this end, freshlyisolated T cells were incubated with 5 μg/ml recombinant humanP-selectin (#ADP3-050, R&D Systems) in RPMI 1640 medium for 15 min at37° C. prior to rolling and adhesion experiments.

The influence of recombinant P-selectin on Blinatumomab-inducedreduction of mean T cell-rolling velocity was evaluated under long-termconditions. While AMG 103 significantly reduced mean T cell-rollingvelocity±SD on HBMEC from 386±77 μm/sec (−AMG 103) to 340±68 μm/sec(+AMG 103) at 45 min after addition of 10 ng/ml Blinatumomab to the flowsystem, further addition of recombinant P-selectin to the experimentreverted this reduction to a mean T cell-rolling velocity±SD of 402±93μm/sec as also observed in the absence of AMG 103 (i.e., −AMG 103, +rec.P-selectin; 384±70 μm/sec). Thus, recombinant P-selectin prevented theBlinatumomab-induced increase of T cell-interactions with endothelialcells (FIG. 16).

Example 3 Doses and Schedules of Co-medications Following the Principleof Anti-leukocyte Adhesion for Mitigating CNS AEs

Administration Schedule for Intravenous PPS Co-Medication withBlinatumomab Treatment for Use in Humans.

Patients receive a 100 mg bolus injection of PPS 3 h±30 min prior tostart of infusion and any dose step of Blinatumomab. Immediately afterthe bolus injection, intravenous administration of PPS is continued byperfusor at 300 mg/day for 72 h.

Administration Schedule for Oral PPS Co-Medication with BlinatumomabTreatment For Use in Humans.

Patients receive 900 mg PPS daily given orally as 300 mg (e.g. 3×100 mg)three times per day starting 7 days before start of infusion and anydose step of Blinatumomab. Oral administration of 900 mg PPS daily iscontinued for 72 h after start of infusion and any dose step ofBlinatumomab.

Administration Schedule for Intravenous Minocycline Co-Medication withBlinatumomab Treatment for Use in Humans.

Patients receive 10 mg/kg Minocycline by short-term intravenous infusion48 h, 24 h and 3 h±30 min prior to and 24 h, 48 h and 72 h after startof infusion and any dose step of Blinatumomab.

Administration Schedule for Oral Minocycline Co-Medication withBlinatumomab Treatment for Use in Humans.

Patients receive 700 mg Minocycline orally once daily 48 h, 24 h and 3h±30 min prior to and 24 h, 48 h and 72 h after start of infusion andany dose step of Blinatumomab.

Administration Schedule for Intravenous Natalizumab Co-Medication withBlinatumomab Treatment for Use in Humans.

Patients receive 300 mg Natalizumab by short-term intravenous infusion24 h prior to start of infusion and any dose step of Blinatumomab.

Example 4 In Vitro Flow Chamber System Using Recombinant AdhesionMolecules

Coating of Recombinant Proteins.

Recombinant proteins (rhICAM-1 #ADP4-050, rhVCAM-1 #ADP5-050 orrhP-selectin #ADP3-050, R&D Systems) were dissolved in ddH₂O accordingto the manufacturer's instructions. Prior to rolling experimentsrecombinant proteins were diluted in Dulbecco's PBS with Ca²⁺ and Mg²⁺(#L1815, Biochrom) and coated onto a μ-slide VI^(0.4) Luer ibiTreat(#80606, ibidi) over night at 4° C. Before usage slides were washedthree times with Dulbecco's PBS and blocked where indicated with 20% FBS(#10270, Gibco) in PBS for 2 h at room temperature. Blocking solutionwas replaced by RPMI 1640 medium (#FG1215, Biochrom) withoutsupplements.

Isolation and Cultivation of Human T Cells.

Isolation and characterization of human peripheral blood mononuclearcells (PBMC) and T cells from blood were performed as described inExample 2 (p. 48, 1-31).

Jurkat E6.1 T cells (#88042803, European Collection of Cell Cultures)were grown at 37° C. and 5% CO₂ in RPMI 1640 medium (#FG1215, Biochrom)containing 10% FBS (#10270, Gibco) and penicillin/streptomycin (#A2213,Biochrom).

For rolling experiments using freshly isolated T cells, these cells wereresuspended in RPMI 1640 medium (#FG1215, Biochrom) without supplementsto a final concentration of 1×10⁶ cells/ml at the start of theexperiment (t=0 min; addition of 10 ng/ml Blinatumomab (AMG 103)) andincubated for 35 min at 37° C. and 5% CO₂. Cells were then filled in aperfusion set “white” (#10963, ibidi) which was connected to both afluidic unit and the μ-slide and image sequences of 45 sec duration wererecorded at t=40 min, 45 min, and 50 min as described in Example 2 (p.50, 18-23).

For rolling experiments using Jurkat E6.1 T cells, these cells wereresuspended in RPMI 1640 medium (#FG1215, Biochrom) without supplementsto a final concentration of 1.5-3×10⁵ cells/ml. Jurkat E6.1 T cells werestarved either over night at 0.5% FBS in RPMI 1640 medium or for 40 minin RPMI 1640 medium without supplements prior to the addition of 10ng/ml Blinatumomab (AMG 103, t=0 min). Cells were incubated for 35 minat 37° C. and 5% CO₂ and then filled in a perfusion set “white” (#10963,ibidi) which was connected to both a fluidic unit and the μ-slide. Imagesequences of 45 sec duration were recorded at t=40 min, 45 min, and 50min as described in Example 2 (p. 50, 18-23).

Incubation of Human T Cells with an Inhibitor of T Cell-SignalTransduction.

For interference with AMG 103-induced adhesion effects, T cells werepre-incubated with a Src kinase inhibitor specifically blocking signaltransduction from the T cell-receptor complex. To this end, theinhibitor PP2 (#529576, Merck, 15 μM) or its vehicle control DMSO(#D2650, Sigma-Aldrich) were added to the T cell-suspension (1×10⁶cells/ml in RPMI 1640 medium) which then was incubated for 40 min at 37°C. and 5% CO₂ prior to addition of 10 ng/ml Blinatumomab (AMG 103, t=0min).

Interaction of Human T Cells with Recombinant Adhesion Molecules UnderFlow Conditions: Assay Setup for Rolling Experiments, Data Acquisitionand Analysis.

Experiments under defined hydrodynamic flow conditions were performed byusing either freshly isolated human T cells or Jurkat E6.1 T cells andμ-slide VI^(0.4) Luer ibiTreat (#80606, ibidi) coated with differentrecombinant proteins as described above. The assay setup was describedin detail in Example 2 (p. 49, 23-37). T cell-rolling experiments wereperformed at a shear stress of 1.1 dyn/cm². T cell-interactions withrecombinant proteins were recorded as described above and data wasanalyzed as described in detail in Example 2 (p. 50, 23-30).

Statistical Data Analysis.

Depending on how many groups of data were compared, an unpaired t-testor a one-way ANOVA combined with a Tukey post-test were used to analyzestatistical significance in Prism (GraphPad Software). A P-value<0.05was regarded as statistically significant. All values are indicated asmean±SD.

Freshly isolated T cells incubated with 10 ng/ml Blinatumomab (+AMG 103)showed a significantly reduced mean T cell-rolling velocity±SD at t=45min on recombinant human ICAM-1 (coated at 12.5 μg/ml, with blocking;−AMG 103: 383±144 μm/sec vs.+AMG 103: 314±79 μm/sec), on recombinanthuman VCAM-1 (coated at 10 μg/ml, with blocking; −AMG 103: 418±136μm/sec vs.+AMG 103: 353±72 μm/sec), and at t=50 min on recombinant humanP-selectin (coated at 20 μg/ml, with blocking; −AMG 103: 402±115 μm/secvs.+AMG 103: 293±73 μm/sec) in semi short-term conditions when comparedto the negative control (−AMG 103) (FIG. 17A-C).

Jurkat E6.1 T cells incubated with 10 ng/ml Blinatumomab (+AMG 103)showed a significantly reduced mean T cell-rolling velocity±SD at t=45min on recombinant human ICAM-1/VCAM-1 (coated at 12.5 and 10 μg/ml,respectively; −AMG 103: 640±91 μm/sec vs.+AMG 103: 518±98 μm/sec), andon recombinant human P-selectin (coated at 20 μg/ml; −AMG 103: 500±81μm/sec vs.+AMG 103: 413±104 μm/sec) in semi short-term conditions whencompared to the negative control (−AMG 103) (FIG. 17D,E).

Thus, Blinatumomab also increases T cell-interactions with recombinanthuman adhesion molecules both for freshly isolated T cells and a commonT cell-line.

The influence of the Src kinase inhibitor PP2 on Blinatumomab-inducedreduction of mean T cell-rolling velocity was evaluated under semishort-term conditions. While AMG 103 in the presence of the vehiclecontrol DMSO significantly reduced mean T cell-rolling velocity±SD onVCAM-1-coated μ-slides (coated at 20 μg/ml) from 259±35 μm/sec (−AMG103) to 205±45 μm/sec (+AMG 103) at t=40 min after addition of 10 ng/mlBlinatumomab, pre-incubation and presence of 15 μM PP2 in the experimentreverted this reduction to a mean T cell-rolling velocity±SD of 256±50μm/sec. PP2 treatment in the absence of AMG 103 resulted in a comparablemean T cell-rolling velocity±SD of 267±66 μm/sec (−AMG 103, +PP2). Thus,Src kinase inhibitor PP2 prevented the Blinatumomab-induced increase ofT cell-interactions with recombinant human VCAM-1 (FIG. 18).

The invention claimed is:
 1. A method of ameliorating, treating, orpreventing a clinical adverse event in a patient caused by administeringblinatumomab to the patient, the method comprising administering to thepatient an effective amount of pentosanpolysulfate (PPS) or apharmaceutically acceptable salt thereof, minocycline, or natalizumab.2. The method according to claim 1, wherein the method further comprisesadministering to the patient genetically engineered T-cells having achimeric antigen receptor (CAR).
 3. The method according to claim 2,wherein said chimeric antigen receptor (CAR) comprises a CD19 bindingdomain.
 4. The method according to claim 1, wherein thepentosanpolysulfate (PPS) or the pharmaceutically acceptable saltthereof, minocycline, or natalizumab is administered prior to orconcomitantly with an initial dosing, a re-exposure, or an increase indose of blinatumomab.
 5. The method according to claim 1, wherein saidclinical adverse event comprises a neurological adverse event.
 6. Themethod according to claim 5, wherein said neurological adverse event isone or more of (i) a cognitive disorder comprising disorientation,confusion or word-finding problems (aphasia), (ii) a seizure, (iii) acerebellar symptom partly observed as an optional prodromal phase of (i)or (ii) comprising a kinetic tremor, ataxia, dysarthria and/orhandwriting problems.
 7. The method according to claim 1, wherein saidpatient is characterized by a B:T-cell ratio of less than 1:5 or aB-cell number of less than about 50 B-cells per μl peripheral blood. 8.The method according to claim 1, wherein said patient is a mammal. 9.The method according to claim 8, wherein said mammal is a primate. 10.The method according to claim 9, wherein said primate is a human.